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Originally published In Press as doi:10.1074/jbc.M503912200 on August 16, 2005

J. Biol. Chem., Vol. 280, Issue 42, 35108-35118, October 21, 2005
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Phosphorylation of Numb Family Proteins

POSSIBLE INVOLVEMENT OF CA2+/CALMODULIN-DEPENDENT PROTEIN KINASES*

Hiroshi Tokumitsu{ddagger}1, Naoya Hatano§, Hiroyuki Inuzuka{ddagger}2, Yuka Sueyoshi{ddagger}, Shigeyuki Yokokura{ddagger}, Tohru Ichimura¶, Naohito Nozaki||, and Ryoji Kobayashi{ddagger}

From the Departments of {ddagger}Signal Transduction Sciences and §Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, the Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, and the ||Department of Biochemistry and Molecular Biology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan

Received for publication, April 11, 2005 , and in revised form, August 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1–293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+/calmodulin-dependent protein kinase I (CaM-KI)3 is a multifunctional CaM-K (reviewed in Refs. 1 and 2) that regulates a wide variety of intracellular Ca2+-signaling pathways, including cell cycle regulation (3–5), myogenesis (6), neurite outgrowth (7), cytoskeletal organization (8), and gene transcription (9, 10). CaM-KI consists of four isoforms ({alpha}, {beta}, {gamma}, and {delta}) derived from different genes; these isoforms are ubiquitously expressed in cells and tissues and are localized primarily in the cytoplasm (11–17). Recent studies have indicated that the CaM-KI{beta} isoforms are localized in both the cytoplasm and nucleus (18) and that the {gamma}-isoforms are anchored to the Golgi and plasma membranes in neurons through a CAAX motif (15, 16). These findings suggest that CaM-KI may play various roles throughout cells (i.e. from the cell membrane to the nucleus). CaM-KI is also conserved among various species, including Aspergillus nidulans (3), Schizosaccharomyces pombe (4), and Caenorhabditis elegans (19).

CaM-KI is part of a protein kinase cascade, referred to as a CaM-K cascade. Full activation of CaM-KI requires the phosphorylation of Thr177 in the activation loop by Ca2+/CaM-dependent protein kinase kinase (CaM-KK) and Ca2+/CaM binding (20–23). It has been shown that Thr177 phosphorylation results in a large increase in the affinity of CaM-KI for its substrate and that Ca2+/CaM binding is required for the release of the autoinhibitory domain from the catalytic core (19, 22, 24). Whereas a number of protein substrates for CaM-KI have been identified (e.g. synapsin I and II (11), the cystic fibrosis transmembrane conductance regulator (26), CREB (27), activating transcription factor (9), and myosin II regulatory light chain (8)), the physiological role of the CaM-KI cascade remains uncertain. To evaluate the physiological functions of the CaM-KK/CaM-KI cascade, it is clearly important to identify the substrates for CaM-KI. A recent study using phosphorylation screening of a cDNA expression library identified novel substrates such as translation initiation factor 4GII; hence, the phosphorylation screening approach may continue to identify additional physiological substrates of CaM-KI (28).

To search for CaM-KI substrates, in this study we developed a functional proteomic method using affinity chromatography with the CaM-KI catalytic domain (CD) as a ligand to partially purify CaM-KI-CD-interacting proteins; we combined this approach with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the identification of substrates. In addition to the identification of two known CaM-KI substrates (synapsin I and CREB) with this method, we identified rat Numbl and Numb, which were bound to the phosphorylated CaM-KI CD at Thr177, and demonstrated that both Numb family proteins are excellent substrates for activated CaM-KI. Furthermore, we characterized the phosphorylation of these Numb family proteins in vitro and in vivo, including their phosphorylation-dependent interactions with 14-3-3 protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—GST-CaM-KI (residues 1–293, K49E) was constructed as previously described (30) and subcloned into the XbaI/XhoI site of the pGEX-KG-PreS vector (31). The recombinant protein was expressed in Escherichia coli JM-109, followed by purification by glutathione-Sepharose chromatography. Recombinant wild-type rat CaM-KI{alpha}, wild-type mouse CaM-KIV, and wild-type rat CaM-KK{alpha} were expressed and purified as previously described (31, 32). Recombinant rat CaM was expressed in the Epicurian coli BL-21 (DE3) using pET-CaM (kindly provided by Dr. Nobuhiro Hayashi, Fujita Health University, Toyoake, Japan) and was purified by phenyl-Sepharose column chromatography (33). The purified catalytic subunit of bovine cAMP-dependent protein kinase (PKA) was kindly provided by Dr. Y. Watanabe (Kagawa University, Kagawa, Japan). Rat CaM-KII holoenzyme was purified from rat forebrain. His-tagged C. elegans CREB (CRH-1{beta}) was expressed and purified as described previously (10). GST-14-3-3{eta} was expressed in E. coli JM-109, followed by purification by glutathione-Sepharose chromatography (51). Anti-phospho-CaM-KI at Thr177 monoclonal antibody was generated as previously described (32). Anti-CaM-KI and anti-CaM-KK antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Transduction Laboratories, respectively. Anti-{alpha}-tubulin, anti-HSP-70, anti-CREB, and anti-synapsin I antibodies were obtained from Amersham Biosciences, MBL International, New England Biolabs, and Chemicon International, respectively. Anti-phospho-CREB antibody was obtained from New England Biolabs. Anti-Numbl and anti-Numb antibodies were purchased from Abcam Ltd. (Cambridge, UK) and Upstate%20Biotechnology">Upstate Biotechnology, Inc. (Lake Placid, NY), respectively. Antibodies to each 14-3-3 isoform were provided by Immuno-Biological Laboratories.

Anti-phospho-Numb/Numbl monoclonal antibody was generated against the synthetic phosphopeptide corresponding to residues 295–314 (CPLEQLVRQGpSFRGFPALSQK; where pS represents phosphoserine) of rat Numbl. The phosphopeptide was conjugated with keyhole limpet hemocyanin via the N terminus cysteine and was injected into Balb/c mice as described previously (34). All other chemicals were obtained from standard commercial sources.

Affinity chromatography—GST-CaM-KI (residues 1–293, K49E) (2.5 mg) was phosphorylated with 3 µg of recombinant rat CaM-KK{alpha} at 30°C for 2 h in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM CaCl2, 1 mM DTT, 2 µg CaM, and 1 mM ATP. Then either phosphorylated or unphosphorylated GST-CaM-KI (residues 1–293, K49E) (2.5 mg) or GST (1 mg) was applied to a glutathione-Sepharose column (250-µl bed volume; Amersham Biosciences), and then the columns were washed with 10 ml of Buffer A (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA). Three fresh whole rat brains and 10 g of fresh rat liver were separately homogenized with 30 ml of Buffer A containing 1 µM microcystin LR and 0.5 µM okadaic acid (Buffer B), followed by centrifugation at 100,000 x g for 30 min. The supernatant (10 ml) was applied to each Sepharose column as described above, followed by washing with 50 ml of Buffer B. The proteins that interacted with GST-CaM-KI (residues 1–293, K49E) were eluted by adding 250 µl of Buffer A containing 30 units of PreScission protease (Amersham Biosciences) to each Sepharose resin, followed by incubation at 4 °C overnight. Twenty-five microliters of SDS-PAGE sample buffer was added to the eluate, and then each sample was stored at –30 °C until analysis by mass spectrometry or Western blotting.

GST-Numb fragment (residues 238–304, 1.5 mg) was phosphorylated with activated CaM-KI (2.1 µg) in the presence of 2 µM CaM and 2 mM ATP in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM CaCl2, 1 mM DTT. Then either phosphorylated or unphosphorylated GST-Numb fragment (residues 238–304) was applied to glutathione-Sepharose columns (250-µl bed volume), and then the columns were washed with 10 ml of Buffer A. Affinity purification of Numb-interacting proteins using the affinity resins in the presence of protein phosphatase inhibitors was performed according to essentially the same protocol as that used for the affinity purification of CaM-KI-interacting proteins described above.

Mass Spectrometry Analysis—A 30-µl sample of the eluate from the unphosphorylated or phosphorylated GST-CaM-KI (residues 1–293, K49E)-coupled glutathione-Sepharose columns described above was separated by SDS-10% PAGE and lightly stained with Coomassie Brilliant Blue. Then 16 gel slices were excised from each sample lane in the ~35–250-kDa range, followed by in-gel digestion with 10 µg/ml trypsin (Promega, Madison, WI) overnight at 37 °C (44). The digested peptides were eluted with 0.1% formic acid and were subjected to LC-MS/MS analysis. LC-MS/MS analysis was performed on a Q-Tof2 quadrupole/time-of-flight hybrid mass spectrometer (Micromass, Manchester, UK) interfaced with capillary reverse-phase liquid chromatography (Micromass CapLCTM system). A 90-min linear gradient from 5 to 45% acetonitrile in 0.1% formic acid was produced and was split at a 1:20 ratio; the gradient solution was then injected into a nano-LC column (PepMap C18, 75 µm x 150 mm; LC Packings, Sunnyvale, CA) at 25 nl/min. The eluted peptides were sprayed directly into the mass spectrometer. The MS/MS data were acquired by MassLynx software (Micromass) and converted to a single text file (containing the observed m/z of the precursor peptide, the fragment ion m/z, and intensity values) by Protein-Lynx software (Micromass). The file was analyzed with the Mascot MS/MS Ions Search (Matrix Science; available on the World Wide Web at www.matrixscience.com) to search and assign the obtained peptides to the NCBI nonredundant data base. We set the search parameters as follows: data base, NCBInr; taxonomy, all; enzyme, trypsin; fixed modifications, carbamidomethyl (C); variable modifications, oxidation (M); peptide tolerance, ±0.2 Da; MS/MS tolerance, ±0.2 Da.

LC-MS/MS was used to identify the phosphorylation site of the Numbl protein, as previously described (35). Two micrograms of phosphorylated recombinant GST-Numbl were separated by SDS-10% PAGE. The following steps were then performed as described above, with two exceptions. First, in-gel digestion was performed with 17 µg/ml chymotrypsin (Roche Applied Sciences) overnight at 25 °C; second, the search parameters were as follows: data base, GST-rat Numbl-Hisx6 (868 amino acid residues); enzyme, all; variable modification, oxidation (M) and phospho-Ser/Thr; peptide tolerance, ±0.1 Da; and MS/MS tolerance, ±0.1 Da.

As regards the identification of Numb-binding proteins, the protein bands of interest were excised from the gel, and in-gel digestion with trypsin (Promega) was performed as described above. The resulting peptides were desalted using a ZipTip µC18 (Millipore Corp.) according to the manufacturer's protocol. Peptides were eluted with 1 µl of matrix ({alpha}-cyano-4-hydroxycinnamic acid) prepared in 50% acetonitrile, 0.1% trifluoroacetic acid, and then the peptides were spotted onto a stainless steel target plate. The protein digests were analyzed by MALDI-TOF MS using the Voyager DE-STR system (Applied Biosystems). Calibration was performed using the autodigestion peaks of trypsin (1045.5642 and 2211.1046). The observed m/z values were submitted to MS-Fit (available on the World Wide Web at prospector.ucsf.edu/ucs-fhtml4.0/msfit.htm) for mass fingerprint searching. We set the search parameters as follows: data base, NCBInr; digest, trypsin; maximum number of missed cleavages, 1; Cys modified by, carbamidomethylation; possible modifications mode, none; minimum number of peptides required to match, 4; mass tolerance, 25 ppm.

Cloning and Construction of Rat Numbl and Numb cDNAs—Rat Numbl cDNA (accession number AB210107 [GenBank] ) was obtained by reverse transcriptase-mediated PCR with Pyrobest DNA polymerase (Takara, Tokyo, Japan) using rat brain cDNA (Invitrogen) as a template (sense primer 5'-CGTCAGATCGAGCCGCCGCCACCACAGCAG-3', derived from the 5'-untranslated region in the cDNA sequence in the data base (XP_218360), and antisense primer 5'-GGCAAGCACAGCATTGGCAGTGAACACAGC-3', derived from the genomic sequence in rat chromosome 1). The initial PCR was followed by a second PCR, using sense primer 5'-GGTCTAGAGATGTCCCGCAGCGCGGCGGCC-3' and antisense primer 5'-GGCTCGAGCTACAGTTCAATCTCGAAGGTC-3' (where underlines indicate nucleotide sequences of restriction sites). Rat Numb cDNA (accession number AB210108 [GenBank] ) was obtained by reverse transcriptase-mediated PCR with Pyrobest DNA polymerase (Takara) using rat brain cDNA (Invitrogen) as a template (sense primer, 5'-CTGTGTCTCCAGGTTGTAAAAGTTAAC-3'; antisense primer, 5'-CCTTGCCTAAGGACAGAAAGAACCATC-3'; both primers were from the genomic sequence in rat chromosome 6). This latter PCR was followed by a second PCR, using sense primer 5'-GGTCTAGACATGAACAAACTACGGCAGAGTTTC-3' and antisense primer 5'-CCCTCGAGCTAAAGCTCTATTTCAAATGCC-3', both of which were derived from the data base (XP_234394 [GenBank] ). The PCR fragments were subcloned into the XbaI/XhoI site of the pGEX-KG-PreS vector. To insert a His6 tag at the C-terminal end of GST-Numbl and also at that of Numb, we inserted annealed oligonucleotides (5'-GAAGTTCTGTTCCAGGGGCCCGAGCACCACCACCACCACCACTGAC-3' and 5'-TCGAGTCAGTGGTGGTGGTGGTGGTGCTCGGGCCCCTGGAACAGAACTTC-3') encoding EVLFQGPEHHHHHH after Leu617 in GST-Numbl (pGEX-KG-PreS-Numbl-His6) or after Leu592 in GST-Numb (pGEX-KG-PreS-Numb-His6). The S304A mutant of pGEX-KG-PreS-Numbl-His6 and the S264A mutant of pGEX-KG-PreS-Numbl-His6 were created by site-directed mutagenesis (GeneEditorTM; Promega) using each mutagenic oligonucleotide. An expression plasmid for GST-fused Numb 238–304 was constructed by PCR using sense primer 5'-GGTCTAGAGACTGCTTCTTTAGAGATGAAC-3' and antisense primer 5'-GGATCCACGCGGAACCAGTGTGTTTTTTATTGGGAA-3', followed by ligation into the XbaI/XhoI sites of pGEX-KG-PreS together with the annealed oligonucleotides encoding the His6 tag, as described above. The C-terminal FLAG-tagged Numbl (pME-Numbl-FLAG) was constructed by PCR using wild type or a S304A mutant Numbl cDNA as a template and antisense primer (5'-CCTCTAGACTACTTATCGTCGTCATCCTTGTAATCCAGTTCAATCTCGAAGGTCTTCTGC-3') containing a region encoding the FLAG epitope, and then the PCR fragment was subcloned into pME18s vector (DNAX Research Institute, Inc.). The nucleotide sequences of all constructs used in this study were confirmed by an ABI377 automated sequencer (PE Biosystems, Foster City, CA).

Purification of GST-Numbl and GST-Numb—cDNAs carrying GST-fused rat Numbl, Numb, and Numb fragment (pGEX-KG-PreS-Numbl-His6, pGEX-KG-PreS-Numb-His6, and pGEX-KG-PreS-Numb 238–304-His6), including S304A (Numbl) and S264A (Numb) mutants, were introduced into E. coli JM-109, and expression of the recombinant proteins was induced by the addition of 1 mM isopropyl-{beta}-D-thiogalactopyranoside. An E. coli pellet containing GST fusion protein was lysed with PBS, followed by purification using glutathione-Sepharose column chromatography, as described in the manufacturer's protocol. Further purification was then carried out by using Ni2+-nitrilotriacetic acid-agarose column chromatography (Qiagen) to obtain GST-fused proteins containing full-length Numbl and Numb. Full-length Numb and Numbl were prepared by the cleavage of GST-Numb and GST-Numbl with PreScission protease.

Phosphorylation of Rat Numbl and Numb in Vitro—Purified recombinant CaM-KI (0.1 mg/ml) or CaM-KIV (0.1 mg/ml) was incubated with or without CaM-KK{alpha} (3 µg/ml) at 30 °C for 20 min in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 2 mM CaCl2, 10 µM CaM, and 200 µM of ATP. The reaction was initiated by the addition of ATP and terminated by a 10-fold dilution with ice-cold 50 mM HEPES (pH 7.5), 2 mg/ml bovine serum albumin, 10% ethylene glycol, and 2 mM EDTA. Small aliquots of CaM-KK-treated (activated) and CaM-KK-untreated (unactivated) CaM-KI or CaM-KIV were stored at –80 °C until used. Purified GST-Numbl, GST-Numb, Numbl, or Numb was incubated without or with unactivated or activated CaM-KI (10 ng) or other protein kinases at 30 °C for the indicated periods in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 2 mM CaCl2, 5 µM CaM, and either 200 µM [{gamma}-32P]ATP (~1000 cpm/pmol) or 200µM ATP for mass spectrometry analysis or Western blotting. Ca2+/CaM was omitted for PKA phosphorylation. The reaction was terminated by the addition of SDS-PAGE sample buffer. The samples were then subjected to SDS-7.5% PAGE followed by either Western blot analysis, autoradiography, or quantification of 32P incorporation into the GST-Numbl by Cerenkov counting of the excised gels.

Phosphorylation of Endogenous Numb in COS-7 Cells—COS-7 cells were maintained in Dulbeco's modified Eagle's medium containing 10% fetal bovine serum. The cells were subcultured in 6-well dishes and then were further cultured in serum-free medium for 22 h, and then the cells were treated with or without 1 µM ionomycin for the indicated period of time. Stimulation was terminated by the addition of 150 µl of SDS-PAGE sample buffer, and then the whole cell lysates were heated at 100 °C for 10 min. After centrifugation, 20 µl of each sample was subjected to SDS-10% PAGE followed by Western blot analysis using antiphospho-Numb/Numbl antibody.

Preparation of Rat Tissue Extract—Rat tissue samples were homogenized with 5 volumes of extraction buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml trypsin inhibitor), and then the samples were centrifuged at 4 °C. SDS-PAGE sample buffer was added to the supernatant, and each sample was stored at –30 °C until used for the Western blot analysis.

Expression of FLAG-tagged Numbl in COS-7 Cells—Transfection of pME-Numbl-FLAG into COS-7 cells was carried out using Lipofectamine reagent (Invitrogen) with 10 µg of the plasmid DNA according to the manufacturer's instruction. After 40 h of incubation, the cells were extracted, and then the cell extract was subjected to the pull-down assay as described below.

Pull-down Assay—Purified GST-14-3-3{eta} (10 µg) was incubated with either phosphorylated or unphosphorylated Numb/Numbl or with COS-7 cell extract containing overexpressed FLAG-tagged Numbl in a solution containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and 10 µg/ml trypsin inhibitor, 1 µM microcystin LR, and 0.5 µM okadaic acid (Buffer C) for 2 h at 4°C or room temperature (for recombinant Numb/Numbl), and then the samples were incubated with 40 µl of glutathione-Sepharose (50% slurry) for 1 h. The glutathione-Sepharose resins were washed five times with buffer C, and then SDS-PAGE sample buffer was added to the resin, followed by either SDS-PAGE or Western blot analysis.

Dephosphorylation of Numbl—GST-Numbl was phosphorylated by activated CaM-KI at 30 °C for 30 min as described above, followed by purification using glutathione-Sepharose column chromatography. Phosphorylated GST-Numbl (1 µg) was either left untreated or incubated with 0.025 units of protein phosphatase 2A (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY) at 30 °C for the indicated period of time in a solution containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT in the presence of either GST (1.5 µg) or GST-14-3-3{eta} (3.2 µg). The reaction was terminated by the addition of SDS-PAGE sample buffer, and the samples were then subjected to SDS-7.5% PAGE, followed by Western blot analysis using either anti-phospho-Numb/Numbl antibody or anti-Numbl antibody.



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  		FIGURE 1.
A functional proteomic approach to identify potential substrates for the CaM-KI cascade. A, schematic representation of affinity ligands coupled with glutathione-Sepharose used for affinity purification of CaM-KI-interacting proteins. Glutathione-Sepharose was coupled with GST (a), unphosphorylated GST-CaM-KI (residues 1–293, K49E) (b), or CaM-KK{alpha}-phosphorylated GST-CaM-KI (residues 1–293, K49E) at Thr177 (c), as described under "Experimental Procedures." Glut., glutathione; CaM-KI cat., CaM-KI catalytic domain (residues 1–293). The cleavage site indicates the location of the recognition sequence (Leu-Glu-Val-Leu-Phe-Gln {downarrow} Gly-Pro) for PreScission protease. B, a protocol of the functional proteomic approach to identify CaM-KI substrates. An equal volume of rat tissue extract was applied to each ligand-coupled glutathione-Sepharose column, as described in A. After the columns were washed, CaM-KI CD-interacting proteins were eluted from each glutathione-Sepharose resin by incubation with 30 units of PreScission protease at 4 °C overnight, followed by Western blot analysis. After each eluted sample was separated by SDS-10% PAGE, 16 gel slices were excised from each sample lane in the ~35–250-kDa range, followed by in-gel digestion with trypsin. The eluted peptides were then analyzed by LC-MS/MS, as described under "Experimental Procedures."

 
Other Methods—Western blot analysis was performed with horse-radish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (Amersham Biosciences) or anti-goat IgG antibody (Sigma) as a secondary antibody, and a chemiluminescence reagent (PerkinElmer Life Sciences) was used for detection. The protein concentration was estimated by staining the samples with Coomassie Brilliant Blue (Bio-Rad) using bovine serum albumin as a standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of CaM-KI Substrates Using a Functional Proteomic Approach—In order to elucidate the physiological significance of CaM-KI, the identification of the target substrates for this enzyme is thought to be extremely important. One of the characteristics of mammalian and C. elegans CaM-KI is that the enzyme is phosphorylated at Thr177 (Thr179 in the C. elegans enzyme) in the activation loop by an upstream activating kinase, CaM-KK, resulting in 20–30-fold increases in the affinity for the substrate peptide (19, 24). Moreover, it has been shown that a mutant of CaM-KI, which has a kinase-negative mutation together with a mutation that disrupts the autoinhibition, functions as a dominant-negative enzyme in transfected cells (7). This evidence indicates that the CD of CaM-KI with a kinase-negative mutation (K49E) could interact with potential substrates. This interaction is expected to be largely induced by phosphorylation at Thr177 by CaM-KK. Thus, the interaction between the Thr177-phosphorylated CaM-KI CD and its substrates could be strong enough to purify target substrates in amounts sufficient for their identification by mass spectrometry. Therefore, we attempted to identify the CaM-KI substrates by combining this affinity chromatography approach that utilizes CaM-KI CD as an affinity ligand with LC-MS/MS analysis.



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  		FIGURE 2.
Analysis of a functional proteomic approach used to identify potential substrates for the CaM-KI cascade from rat brain extract. Rat brain extract was subjected to the functional proteomic analysis as described in the legend to Fig. 1. Five microliters of eluted sample from glutathione-Sepharose coupled with GST (lane a), unphosphorylated GST-CaM-KI (residues 1–293, K49E, lane b), or Thr177-phosphorylated GST-CaM-KI (residues 1–293, K49E, lane c) were subjected to SDS-10% PAGE, followed by either Coomassie Brilliant Blue (CBB) staining (A, left) or Western blotting using antiphospho-CaM-KI antibody (0.5-µl sample; A, right), anti-{alpha}-tubulin antibody (B, left), anti-HSP-70 antibody (B, right), anti-CREB antibody (C, left), or anti-synapsin I antibody (C, right). The arrow indicates the cleaved CaM-KI CD.

 
As shown in Fig. 1A, we constructed and purified a GST-fused CaM-KI CD (residues 1–293, K49E) for the affinity ligand. This GST-CaM-KI CD had a cleavage site (Leu-Glu-Val-Leu-Phe-Gln {downarrow} Gly-Pro) for PreScission protease between GST and the CaM-KI CD, which led to the specific elution of the CaM-KI CD-interacting proteins from the affinity matrix by protease treatment. GST-fused CaM-KI CD was fully phosphorylated with CaM-KK in the presence of Ca2+/CaM for 2 h, as was recently demonstrated (32), and then phosphorylated CaM-KI CD was coupled with glutathione-Sepharose resin (Fig. 1A, c). As controls, we prepared two glutathione-Sepharose columns, one coupled with GST alone (Fig. 1A, a) and the other with unphosphorylated GST-fused CaM-KI CD (Fig. 1A, b). An equal volume of rat brain extract was applied to each column in the presence of protein phosphatase inhibitors (microcystin LR and okadaic acid), followed by extensive washing of each column (Fig. 1B). CaM-KI CD-interacting proteins were eluted by incubation with 30 units of PreScission protease at 4 °C overnight. The same volume of eluate from each column was subjected to SDS-PAGE, followed by either protein staining (Fig. 2A, left) or Western blot analysis with anti-phospho-CaM-KI antibody (Fig. 2A, right). The Thr177-phosphorylated CaM-KI CD (lane c) and the unphosphorylated CaM-KI CD (lane b) were cleaved from GST and were eluted from each column in approximately the same amounts, together with the CaM-KI CD-associated proteins with a molecular weight range of ~40–250 kDa (Fig. 2A, left). The proteins eluted from the columns, together with the cleaved CaM-KI CD, appeared to interact directly with CaM-KI CD, since we did not detect any proteins with a molecular mass higher than 30 kDa in the eluate from the GST-coupled glutathione-Sepharose (Fig. 2A, left, lane a). Next, to identify the CaM-KI CD-interacting proteins, we excised 16 gel slices in the ~35–250-kDa range from the SDS-polyacrylamide gel, in which the eluates from either Thr177-phosphorylated or unphosphorylated CaM-KI CD-coupled glutathione-Sepharose were separated. The slices were then subjected to in-gel digestion with trypsin. The digested peptides eluted from each slice were subjected to LC-MS/MS analysis in order to identify the proteins by searching the NCBI data base. Among the proteins we detected from the Thr177-phosphorylated and unphosphorylated CaM-KI CD-coupled glutathione-Sepharose samples, ~100 were found to occur in both samples. These proteins included {alpha}-tubulin and HSP-70, which have been found to possess the assigned peptides in similar numbers (data not shown). We confirmed by Western blot analysis that approximately the same amounts of {alpha}-tubulin (Fig. 2B, left) and HSP-70 (Fig. 2B, right) were eluted from both Thr177-phosphorylated and unphosphorylated CaM-KI-CD-coupled resins, thus indicating that both proteins interacted with CaM-KI CD, at least in a Thr177 phosphorylation-independent manner. However, we detected a large amount of synapsin I in the eluate from Thr177-phosphorylated CaM-KI CD-coupled glutathione-Sepharose (the assigned peptides covered 48% of the total sequence of rat synapsin I), in contrast to that detected in the eluate from unphosphorylated CaM-KI CD-coupled resin (the assigned peptides covered 9% of the total sequence of rat synapsin I) (data not shown). Western blot analysis with anti-synapsin I antibody clearly indicated that CaM-KK-mediated Thr177 phosphorylation significantly increased the amount of synapsin I available to interact with CaM-KI CD (Fig. 2C, right). In addition, we detected two peptides derived from rat CREB (residues 136–150, KILNDLSSDAPGVPR; residues 137–150, ILNDLSSDAPGVPR), but only in the eluate from Thr177-phosphorylated CaM-KI CD-coupled glutathione-Sepharose. This finding is consistent with the results of the Western blot analysis using anti-CREB antibody (Fig. 2C, left), namely, CREB was detected only in the sample from Thr177-phosphorylated CaM-KI CD-coupled resin (lane c). Synapsin I and CREB have been shown to be phosphorylated in vitro by CaM-KI at Ser9 (P-site 1) and Ser133, respectively (27, 36).



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  		FIGURE 3.
Identification of rat Numbl as a potential substrate for the CaM-KI cascade. A, a comparison of the amino acid sequences for rat Numbl and Numb proteins as deduced from cDNAs isolated in this study. Identical residues are indicated by an asterisk. The amino acid sequences of the peptides (boldface type) from either rat Numbl or Numb, eluted from Thr177-phosphorylated GST-CaM-KI (residues 1–293, K49E)-coupled glutathione-Sepharose, were determined by LC-MS/MS, as described in the legend to Fig. 1 and under "Experimental Procedures." The phosphorylation site of either rat Numbl (Ser304) or Numb (Ser264) determined in this study is indicated by a solid bar. The PTB domain is indicated by a box. The fragment of rat Numb (residues 238–304) used for the identification of the Numb binding proteins (as shown in Fig. 8) is underlined. B, eluted sample (5 µl) using rat brain extract from glutathione-Sepharose resin coupled with GST (lane a), unphosphorylated GST-CaM-KI (residues 1–293, K49E, lane b), or Thr177-phosphorylated GST-CaM-KI (residues 1–293, K49E, lane c), as shown in Fig. 2, was subjected to Western blot analysis using anti-Numbl antibody. The arrow indicates rat Numbl. The asterisk indicates eluted CaM-KI (residues 1–293, K49E) ligands, which were nonspecifically bound to the secondary antibody.

 
Identification of Numbl from Rat Brain as a CaM-KI Substrate—In addition to identifying two known substrates for CaM-KI (synapsin I and CREB), we obtained seven peptides derived from Numbl (Fig. 3A, boldface sequences in rat Numbl) only in the eluate from Thr177-phosphorylated CaM-KI CD-coupled affinity resin. Numbl is known to belong to the Numb family of proteins, which suppress Notch signaling during the determination of cell fate in the context of neuronal development; this effect possibly occurs via the direct interaction between Numb family proteins with the intracellular domain of Notch (37–39). Recent studies have demonstrated that Numb and Numbl play redundant but crucial roles in maintaining neural progenitor cells during mouse neurogenesis (40, 41). In the present study, specific binding of rat Numbl to Thr177-phosphorylated CaM-KI CD was clearly observed by Western blotting with anti-Numbl antibody (Fig. 3B); this result is similar to results observed with CREB and synapsin I (Fig. 2C). Whereas all seven of the peptides obtained were completely matched with those from mouse and human Numbl (39), one peptide (residues 415–424; WLEEVSQVAK) did not match a corresponding sequence (residues 416–425; WLEEVSQPPF) in rat Numbl in the data base (accession number XP_218360). This finding indicates that the Numbl that we identified from rat brain tissue is a product of a splice variant derived from the Numbl gene. Therefore, we attempted reverse transcription-PCR cloning of Numbl cDNA obtained from rat brain tissue, as described under "Experimental Procedures." The deduced amino acid sequence (617 amino acid residues) obtained from rat Numbl cDNA (1854 bp; accession number AB210107 [GenBank] ) contained all seven of the peptide sequences we had determined by LC-MS/MS (Fig. 3A). The rat Numbl we obtained contains a 29-poly-Gln repeat (residues 426–454) derived from a CAG repeat, which is a characteristic of the Numbl protein in various species, as well as a phosphotyrosine binding (PTB; residues 65–204) domain (39, 42).



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  		FIGURE 4.
Phosphorylation of rat Numbl at Ser304 by the CaM-KI cascade in vitro. A, wild-type (WT) or an S304A mutant (SA) of purified GST-Numbl (2 µg) was incubated without (CaM-KK–, CaM-KI–), or with either unactivated CaM-KI (10 ng; CaM-KK–, CaM-KI+) or activated CaM-KI (10 ng; CaM-KK+, CaM-KI+) at 30 °C for 5 min in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 2 mM CaCl2, 5 µM CaM, and 200 µM [{gamma}-32P]ATP as described under "Experimental Procedures." The reaction was terminated by the addition of SDS-PAGE sample buffer. The samples were then subjected to SDS-7.5% PAGE followed by either protein staining (upper panel) or autoradiography (lower panel). Lane M in the upper panel shows the molecular weight marker. B, GST-Numbl, which was phosphorylated by activated CaM-KI for 5 min (as shown in A) was subjected to SDS-PAGE, digested with chymotrypsin, and then analyzed by LC-MS/MS to identify phosphoserine 304. The singly charged ion of a peptide (residues 297–305) derived from phosphorylated GST-Numbl was subjected to MS/MS analysis as described under "Experimental Procedures." The observed y-ion and b-ion fragment series generated by collision-induced dissociation are indicated by arrows. The observed fragment ions are indicated above and below the peptide sequence. Labeled peaks without arrows of m/z 121.04, 240.10, and 353.18 were assigned as (b2-NH3)/2, b2-H2O, and b3-H2O, respectively. C, time course experiment of Numbl phosphorylation. Wild-type GST-Numbl (2 µg) was incubated with activated CaM-KI (10 ng) in the presence of 200 µM [{gamma}-32P]ATP at 30 °C for the indicated period of time, as described in A. Phosphorylated GST-Numbl was subjected to SDS-10% PAGE, followed by protein staining (inset, top) or autoradiography (inset, bottom). 32P incorporation into GST-Numbl was quantified by Cerenkov counting of the excised gels.

 
Next, we examined whether rat Numbl could be phosphorylated by CaM-KI in vitro. For this purpose, we constructed and purified GST-Numbl, in which a His6 tag was inserted at the C-terminal end to obtain a GST-fused protein containing full-length Numbl. When GST-Numbl (2 µg) was incubated in the presence of Ca2+/CaM and [{gamma}-32P]ATP at 30 °C for 5 min without or with either unactivated or activated CaM-KI (10 ng) having a CaM-KK phosphorylation at Thr177, only the activated CaM-KI efficiently phosphorylated GST-Numbl (Fig. 4A). This finding was in good agreement with results demonstrating that Numbl can directly interact only with Thr177-phosphorylated CaM-KI CD and not with unphosphorylated CaM-KI CD (Fig. 3B). To determine the site(s) of Numbl that are phosphorylated by activated CaM-KI, we analyzed phosphorylated GST-Numbl (2 µg) as described above by in-gel digestion with chymotrypsin, followed by LC-MS/MS analysis. We obtained peptide sequences that covered 78% of the entire amino acid sequence of Numbl (data not shown). Among these peptides, we detected a single phosphopeptide corresponding to residues 297–305 in Numbl. LC-MS/MS analysis also revealed a single phosphorylation site at Ser304 in the peptide (Fig. 4B). To confirm the phosphorylation of Numbl at Ser304 by activated CaM-KI, we used a GST-Numbl S304A mutant in a phosphorylation assay (Fig. 4A, right lane). It was observed that 32P incorporation into GST-Numbl S304A was significantly lower than that into the wild-type Numbl, although weak residual phosphorylation was observed in the mutant. These results indicate that the Ser304 residue in rat Numbl is a primary site for phosphorylation by CaM-KK-activated CaM-KI in vitro. The Ser304 in rat Numbl is conserved in mouse and human counterparts as well as in Numb (Ser264), another Numb family protein (Fig. 3A). Next, we kinetically analyzed rat Numbl phosphorylation (Fig. 4C). A time course experiment on the phosphorylation of GST-Numbl (2 µg) by activated CaM-KI (10 ng) revealed that rat Numbl was rapidly (t1/2 = 1–2 min) and stoichiometrically (~0.9 mol of Pi incorporation into 1 mol of GST-Numbl) phosphorylated under these conditions.

Identification of Numb from Rat Liver as a CaM-KI Substrate—In a separate experiment employing this proteomic approach as applied to rat liver extract, four peptides derived from rat Numb (Fig. 3A, bold-faced sequences in rat Numb) were detected; however, these peptides were only detected in the eluate from Thr177-phosphorylated CaM-KI CD-coupled glutathione-Sepharose. This finding, which was supported by the results of Western blot analysis using anti-Numb antibody (Fig. 5A, right), was also similar to what we observed with Numbl from rat brain extract (Fig. 3B). We detected two immunoreactive bands with molecular masses of ~75 and ~65 kDa, which was consistent with a previous report in which four mouse Numb isoforms having molecular masses of 65, 66, 71, and 72 kDa were generated by alternative splicing of the Numb mRNA (43). CREB from rat liver was also detected only in the eluate from Thr177-phosphorylated CaM-KI CD-coupled resin (Fig. 5A, left panel). These results suggest that both Numbl and Numb are potential substrates for activated CaM-KI. Therefore, we cloned Numb cDNA (1779 bp; accession number AB210108 [GenBank] ) by reverse transcription-PCR, which revealed that this cDNA encoded a p65 isoform composed of 592 amino acid residues (Fig. 3A) (43) and then produced recombinant GST-fused Numb protein. Similar to the results of the phosphorylation experiment with rat Numbl (Fig. 4A), GST-fused Numb was significantly phosphorylated, but only by activated CaM-KI, and 32P-incorporation into rat Numb was largely reduced by an Ala mutation at Ser264 (Fig. 5B).



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  		FIGURE 5.
Identification of rat Numb as a potential substrate for the CaM-KI cascade. A, rat liver extract was subjected to the functional proteomic method, as described in Fig. 1. Five microliters of eluted sample using rat liver extract from glutathione-Sepharose resin coupled with GST (lane a), unphosphorylated GST-CaM-KI (residues 1–293, K49E, lane b), or Thr177-phosphorylated GST-CaM-KI (residues 1–293, K49E, lane c) were subjected to Western blot analysis using either anti-CREB antibody (left) or anti-Numb antibody (right). B, phosphorylation of rat Numb at Ser264 by the CaM-KI cascade. Wild-type (WT) or a S264A mutant (SA) of purified GST-Numb (1µg) was incubated without (CaM-KK–, CaM-KI–) or with either unactivated CaM-KI (10 ng; CaM-KK–, CaM-KI+) or activated CaM-KI (10 ng; CaM-KK+, CaM-KI+) in the presence of 200 µM [{gamma}-32P]ATP at 30 °C for 5 min as described in Fig. 4A. The reaction was terminated by the addition of SDS-PAGE sample buffer. The samples were then subjected to SDS-7.5% PAGE, followed by either protein staining (top) or autoradiography (bottom).

 
Characterization of Phosphorylation of Numb and Numbl by Antiphospho-Numb/Numbl Antibody—To examine the phosphorylation of Numb family proteins in vivo, we produced a monoclonal antibody that specifically recognized the phosphorylated form of Numb at Ser264 as well as that of Numbl at Ser304. This antibody specifically recognized both Numbl (Fig. 6A, left) and Numb (Fig. 6B, left) phosphorylated by activated CaM-KI at 30 °C for 20 min to at least the nanogram level of the phosphoproteins. However, the antibody did not recognize either the unphosphorylated forms or the mutants that had an Ala substitution at the phosphorylation sites. This phosphospecific antibody allowed us to determine whether or not the partially purified rat tissue-derived Numbl and Numb specifically bound to Thr177-phosphorylated CaM-KI CD were phosphorylated at each CaM-KI phosphorylation site. Here (Fig. 6, A and B, left panels, lane c), we did not observe any phosphorylation at Ser304 and Ser264 in Numbl and Numb, respectively, from rat tissues bound to Thr177-phosphorylated CaM-KI CD, whereas the amounts of both proteins tested were comparable with those of recombinant phospho-Numb family proteins (Fig. 6, A and B, right panels). This result clearly demonstrated that Thr177-phosphorylated CaM-KI CD specifically interacted with the unphosphorylated substrates.

Since multifunctional CaM-Ks (including CaM-KI, CaM-KII, and CaM-KIV) have been shown to phosphorylate similar consensus sequences, we examined whether two other CaM-Ks (CaM-KII and CaM-KIV) were capable of phosphorylating the CaM-KI-sites of Numbl (Ser304) and Numb (Ser264). We also investigated the phosphorylation of Numbl by PKA. As a control experiment (Fig. 6C, left), we confirmed that all of the kinases tested were able to phosphorylate the Ser29 residue of C. elegans CREB protein (CRH-1{beta}) (10). In addition to activated CaM-KI, we found that CaM-KII and activated CaM-KIV by CaM-KK phosphorylation were capable of phosphorylating the Ser304 residue of Numbl as well as the Ser264 residue of Numb; however, PKA did not phosphorylate these residues (Fig. 6C, right two panels).



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  		FIGURE 6.
Characterization of the phosphorylation of rat Numb and Numbl by anti-phospho-Numb/Numblantibody. A, wildtype (WT) or a S304A mutant (SA) of GST-Numbl was either treated with activated CaM-KI (10 ng) in the presence of 200 µM ATP at 30 °C for 20 min as described in Fig. 4A (+) or left untreated (–), and then the indicated amount of protein was subjected to Western blot analysis using either anti-phospho-Numb/Numbl antibody (left) or anti-Numbl antibody (right). Partially purified Numbl (5-µl sample) from rat brain extract with Thr177-phosphorylated GST-CaM-KI (residues 1–293, K49E)-coupled resin (as shown in Fig. 3B) was also analyzed (lane c). B, wild type (WT) or an S264A mutant (SA) of GST-Numb was either treated with activated CaM-KI in the presence of 200 µM ATP at 30 °C for 20 min as described in Fig. 4A (+) or left untreated (–), and then the indicated amount of protein was subjected to Western blot analysis using either anti-phospho-Numb/Numbl antibody (left) or anti-Numb antibody (right). Partially purified Numb (5-µl sample) from rat liver extract with Thr177-phosphorylated GST-CaM-KI (residues 1–293, K49E)-coupled resin (as shown in Fig. 5A) was also analyzed (lane c). C, phosphorylation of Numbl and Numb by multifunctional CaM-Ks. GST-Numbl (right upper panel), GST-Numb (right lower panel), or C. elegans CREB (CRH-1{beta}; left panel) was incubated without (None) or with PKA (PKA), activated CaM-KI (CaM-KI), CaM-KII (CaM-KII), or activated CaM-KIV (CaM-KIV)(~10 ng of enzyme) in the presence of Mg-ATP and Ca2+/CaM (for CaM kinases) at 30 °C for 10 min, followed by Western blot analysis using anti-phospho-CREB antibody (left) or anti-phospho-Numb/Numbl antibody (right). The asterisk indicates phospho-CRH-1{beta}.

 



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  		FIGURE 7.
Phosphorylation of Numb family proteins in rat tissues and in COS-7 cells. A, rat tissue extracts (30 µg) were subjected to Western blot analysis using anti-phospho-Numb/Numbl antibody. B, COS-7 cells were cultured in serum-free medium for 22 h and then were treated without or with 1µM ionomycin for the indicated periods of time (2–20 min). Stimulation was terminated by the addition of 150 µl of SDS-PAGE sample buffer. Then the whole cell lysates (20 µl) were subjected to Western blot analysis using anti-phospho-Numb/Numbl antibody (middle panel). The COS-7 cell lysates were analyzed by Western blotting using anti-CaM-KI antibody (lane a), anti-CaM-KK antibody (lane b), or anti-Numb antibody (lane c). C, the phosphorylation of Numb in COS-7 cells (as shown in B) was quantified by densitometric scanning of 65-kDa immunoreactive signals.

 
Phosphorylation of Numb in Vivo and in Intact Cells—In order to examine whether or not Numb family proteins are phosphorylated at the CaM-KI phosphorylation site in vivo, we analyzed various rat tissue extracts by Western blot analysis using the phosphospecific antibody (Fig. 7A). We detected the phosphorylation of a 65-kDa Numb family protein in all of the tissue extracts tested, along with the weak phosphorylation of a 75-kDa Numb family protein in several tissues, indicating that the Numb family proteins are phosphorylated in vivo. However, we were unable to distinguish between the phosphorylation of Numbl and Numb isoforms (43) in the crude tissue extract, due to the similarity of the immunoreactivity of the antibody against phospho-Numb and phospho-Numbl (Fig. 6, A and B). Therefore, to examine the phosphorylation of Numb family proteins in intact cells, we decided to use COS-7 cells, since we readily detected two Numb isoforms with molecular masses of ~75 and ~65 kDa in these cells (Fig. 7B, lane c); this result was similar to that observed with partially purified Numb isoforms from rat liver extract (Fig. 5A, right, lane c). We also detected CaM-KK and CaM-KI in COS-7 cells (Fig. 7B, lanes a and b). We stimulated COS-7 cells with 1 µM ionomycin for various periods of time after 22-h serum starvation, and then the whole cell lysates were analyzed by Western blotting using the anti-phospho-Numb/Numbl antibody (Fig. 7, B (middle) and C). The immunoreactivity of the 65-kDa protein, which was possibly a phosphorylated form of the Numb isoform, was detected in the sample without stimulation. The phosphorylation of the 65- and 75-kDa Numb isoforms was rapidly induced (~2.5-fold) in response to ionomycin treatment. Phosphate incorporation peaked within 2–5 min and then gradually decreased to the basal level within 15 min; this decrease might have been due to the enhanced dephosphorylation of Numb. We obtained a similar result with HeLa cells, indicating that endogenous Numb is phosphorylated at Ser264 and that phosphorylation is induced in response to intracellular Ca2+ mobilization in intact cells.

CaM-KI Phosphorylation of Numb Family Proteins Regulates Interaction with 14-3-3 Proteins—To further our understanding of the functional consequences of Numb/Numbl phosphorylation, we attempted to identify the proteins that interact with Numb/Numbl in a phosphorylation-dependent manner. To purify the Numb-interacting proteins from rat liver extract, we used as the affinity ligands an unphosphorylated GST-fused Numb fragment (residues 238–304; underlined sequence in Fig. 3A) and a GST-fused Numb fragment phosphorylated at Ser364 by activated CaM-KI. The Numb-interacting proteins were eluted by treatment of the affinity resins with PreScission protease to cleave the Numb fragment from GST according to essentially the same protocol as that used for the affinity purification of CaM-KI-interacting proteins, followed by SDS-PAGE analysis (Fig. 8A, left panel) and Western blotting with anti-phospho-Numb/Numbl antibody (Fig. 8A, right panel). As shown in Fig. 8A, proteins with molecular masses of 32 and 30 kDa were purified specifically with phosphorylated GST-Numb fragment (lane b). Mass fingerprinting identified those Numb-binding proteins as 14-3-3{epsilon} (32-kDa band), 14-3-3{theta}, and 14-3-3{gamma} (30-kDa bands), respectively, indicating that the 14-3-3 isoforms were capable of interacting with the Numb fragment (residues 238–304) in a phosphorylation-dependent manner. To examine whether the Numb fragment preferentially binds to any specific 14-3-3 isoform(s), we analyzed purified samples by Western blotting using isoform-specific anti-14-3-3 antibodies (Fig. 8B). Here we found that all of the 14-3-3 isoforms ({beta}, {gamma}, {epsilon}, {zeta}, {eta}, and {theta}) tested interacted with the Ser264-phosphorylated Numb fragment. We then investigated whether the full-length Numb protein could interact with 14-3-3 protein in a phosphorylation-dependent manner by using a pull-down assay. The GST-14-3-3{eta} was incubated with recombinant Numb with or without activated CaM-KI phosphorylation, and then the samples were subjected to the pull-down assay with glutathione-Sepharose, followed by Western blot analysis using anti-Numb antibody (Fig. 8C). As a result, the phosphorylated form of Numb was found to specifically interact with GST-14-3-3{eta}. We then examined the phosphorylation-dependent interaction of Numbl with 14-3-3 protein by pull-down assay using recombinant Numbl expressed in E. coli (Fig. 9A) and COS-7 cells extract containing overexpressed FLAG-tagged Numbl (Fig. 9B). The results clearly demonstrated that the 14-3-3{eta} protein interacted with phosphorylated Numbl at Ser304 but not with unphosphorylated Numbl or with the S304A mutant treated with activated CaM-KI (Fig. 9A). These findings were confirmed with C-terminal FLAG-tagged Numbl expressed in COS-7 cells. A certain population of FLAG-tagged Numbl was phosphorylated at Ser304 in transfected COS-7 cells (Fig. 9B, top two panels), which was consistent with our observation regarding the phosphorylation of endogenous Numb in nonstimulated COS-7 cells (Fig. 7B). We found that the phosphorylated form of FLAG-tagged wild-type Numbl was specifically pulled down with GST-14-3-3{eta}, whereas no interaction of the Numbl S304A mutant with GST-14-3-3{eta} was observed (Fig. 9B, bottom two panels), thus indicating that the phosphorylation of Ser304 is critical for the association of Numbl with 14-3-3 protein. Finally, we tested whether the interaction with 14-3-3 protein might regulate dephosphorylation of Numbl. With activated CaM-KI-phosphorylated GST-Numbl as substrate, protein phosphatase 2A gave robust dephosphorylation of Ser304, and this dephosphorylation was completely blocked by the presence of GST-14-3-3{eta} (Fig. 9C).



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  		FIGURE 8.
Identification of 14-3-3 proteins as Numb-interacting proteins. A, rat liver extract was subjected to affinity chromatography using GST-Numb fragment (residues 238–304) and activated CaM-KI-phosphorylated GST-Numb fragment (residues 238–304) as affinity ligands, and then the Numb-binding proteins were eluted by the cleavage of the Numb fragments from GST with PreScission protease treatment, as described under "Experimental Procedures." Ten microliters of eluted sample from glutathione-Sepharose resin coupled with either GST-Numb fragment (lane a) or phosphorylated GST-Numb fragment (lane b) was subjected to SDS-15% PAGE, followed by protein staining (left) or Western blot analysis using anti-phospho-Numb/Numbl antibody (1-µl sample, right panel). Eluted proteins from phosphorylated GST-Numb fragment-coupled resin (lane b) with molecular masses of 32 and 30 kDa were identified by mass finger-printing as the 14-3-3{epsilon}, 14-3-3{theta}, and 14-3-3{gamma} isoforms, respectively, as described under "Experimental Procedures." B, eluted proteins (1-µl sample) from phosphorylated GST-Numb fragment-coupled resin as in A, lane b, were analyzed using antibodies to 14-3-3{beta}, 14-3-3{gamma}, 14-3-3{epsilon}, 14-3-3{zeta}, 14-3-3{eta}, and 14-3-3{theta} isoforms. C, recombinant rat Numb was either treated with activated CaM-KI (10 ng) in the presence of 200 µM ATP at 30 °C for 20 min as described in Fig. 4A (+) or left untreated (–) and then was subjected to a pull-down assay with GST-14-3-3{eta}, followed by Western blot analysis using anti-Numb antibody (right) as described under "Experimental Procedures." The left panel shows the input Numb proteins examined with anti-Numb antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated a novel functional proteomic approach to identify potential substrates for the CaM-KI cascade using mass spectrometry analysis. Here, we have shown that the CaM-KI CD efficiently trapped two known CaM-KI substrates (CREB and synapsin I) in a Thr177 phosphorylation-dependent manner. In addition, we identified two Numb family proteins (Numb and Numbl) from rat tissues as novel substrates for activated CaM-KI. The present affinity chromatography approach was used to partially purify Numbl and Numb from rat tissues; here both Numbl and Numb were found to exhibit dephosphorylated forms, indicating that the Thr177-phosphorylated CaM-KI CD specifically recognizes and traps unphosphorylated substrates, but the unphosphorylated CaM-KI CD is unable to trap such substrates. This observation directly demonstrates that the phosphorylation of the activation loop Thr residue significantly induces a physical interaction between the CaM-KI CD and its potential substrates. Therefore, Thr177 phosphorylation by CaM-KK appears to be crucial for the ability of CaM-KI to recognize and phosphorylate its substrates. In a manner similar to that of CaM-KI, the activity of many protein kinases is regulated by the phosphorylation of the activation loop Thr residue, often by an upstream kinase (29, 47). A phosphate on the activation-loop properly positions the loop for both catalysis and substrate recognition. For example, it has been demonstrated that CDK2 (48), ERK2 (49), and CaM-KIV (50) enhance their affinity for substrates by activation loop phosphorylation. Therefore, our functional proteomic approach using the activation loop phosphorylated CD of the protein kinase as an affinity ligand could be applicable for the identification of potential substrates of relevant kinases.



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  		FIGURE 9.
Phosphorylation-dependent interaction of 14-3-3 protein with Numbl. A, recombinant rat Numbl (wild-type (WT) or S304A mutant (SA)) was either treated with activated CaM-KI (10 ng) in the presence of 200 µM ATP at 30 °C for 20 min as described in Fig. 4A (+) or left untreated (–) and then was subjected to a pull-down assay with GST-14-3-3{eta} (Pull-down). The samples were then subjected to SDS-10% PAGE, followed by either protein staining (upper and middle panels) or Western blot analysis using antiphospho-Numb/Numbl antibody (lower panel). The upper panel (Input) shows the input Numbl proteins examined by protein staining. The arrows indicate Numbl, and the asterisk indicates GST-14-3-3{eta}. B, extract of COS-7 cells expressing either wild type (WT) or an S304A mutant (SA) of C-terminal FLAG-tagged Numbl (Numbl-FLAG) was subjected to a pull-down assay with GST-14-3-3{eta} (Pull-down), followed by Western blot analysis (WB) using anti-FLAG antibody ({alpha}-FLAG) or anti-phospho-Numb/Numbl antibody ({alpha}-Phospho-Numb/Numbl) as described under "Experimental Procedures." The input lysates (upper two panels, Input) were examined with anti-FLAG antibody to confirm the equal expression of the Numbl-FLAG constructs or with anti-phospho-Numb/Numbl antibody ({alpha}-Phospho-Numb/Numbl) to verify the phosphorylation of Numbl-FLAG. C, dephosphorylation of Numbl by protein phosphatase 2A. Phosphorylated GST-Numbl (1 µg) was either left untreated (–) or incubated with 0.025 units of protein phosphatase 2A at 30 °C for the indicated times in the presence of either GST (1.5 µg; +GST) or GST-14-3-3{eta} (3.2 µg; +GST-14-3-3{eta}) as described under "Experimental Procedures." The reaction was terminated by the addition of SDS-PAGE sample buffer, and the samples were then subjected to Western blot analysis using either anti-phospho-Numb/Numbl antibody ({alpha}-Phospho-Numb/Numbl) or anti-Numbl antibody ({alpha}-Numbl). The arrows indicate GST-Numbl. Similar results were obtained for at least three independent experiments.

 
Numb family proteins have been demonstrated to play redundant but critical roles in cell fate determination, especially as regards the maintenance of neural progenitor cells during neurogenesis (37–41). However, neither the regulation of Numb/Numbl function by phosphorylation nor the cross-talk of Numb/Numbl with Ca2+ signaling has been explored. In addition to the identification of equivalent phosphorylation sites in both Numb at Ser264 (in p65 isoform) and Numbl at Ser304 by activated CaM-KI, we detected the site-specific phosphorylation of Numb family proteins in various rat tissue extracts and cultured cells, supporting the hypothesis that the phosphorylation of Numb family proteins is physiological. Thus, the present finding may shed light on the dynamic regulation of Numb/Numbl activity by phosphorylation.

The primary sequences surrounding the CaM-KI phosphorylation sites of both Numb family proteins (Leu-Val-Arg-Gln-Gly-Ser(P)304-Phe-Arg-Gly-Phe in Numbl and Leu-Ala-Arg-Gln-Gly-Ser(P)264-Phe-Arg-Gly-Phe in Numb) match completely with an optimal consensus sequence for substrate recognition of CaM-KI (Hyd-Xaa-Arg-Xaa-Xaa-Ser(P)/Thr(P)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue), as determined by synthetic peptides (25). This is consistent with the notion that Numb family proteins are the down-stream targets of the Ca2+/CaM-dependent signaling cascade, and CaM-KK/CaM-KI is implicated in this pathway. However, we also found that two other multifunctional CaM-Ks (CaM-KII and CaM-KIV) were capable of phosphorylating the CaM-KI phosphorylation sites in both Numbl and Numb in vitro. This observation suggested the possibility that multiple CaM kinases might be involved in enhanced Numb phosphorylation in response to the Ca2+ mobilization observed in COS-7 cells; this possibility will need to be addressed in the future.

Numb family proteins are known to structurally resemble an adaptor or scaffold protein that contains a PTB domain (42), a proline-rich carboxyl terminal region containing several putative Src homology 3 domain-binding sites (45), and an Eps15 homology domain-binding motif (46). Although the middle portions surrounding the CaM-KI phosphorylation sites (residues 288–347 in rat Numbl and residues 248–307 in rat Numb) (Fig. 3A) exhibit high homology in Numb and Numbl (~87% identity), the function of this region has not yet been elucidated. Determination of phosphorylation in this conserved region in vitro and in vivo suggested that the middle portions of Numb/Numbl might exert similar function(s), such as protein/protein interaction, which could in turn be regulated by phosphorylation. Indeed, we demonstrated that 14-3-3 proteins were capable of interacting with Numb and Numbl in a phosphorylation-dependent manner at Ser264 and Ser304, respectively. Based on our results, it appears that one of the effects of 14-3-3 protein binding to phosphorylated Numb family proteins may be to block subsequent dephosphorylation, resulting in the maintenance of Numb/Numbl in a phosphorylated state. The 14-3-3-interaction region containing the CaM-KI phosphorylation site is located adjacent to the PTB domain of Numb/Numbl, which has been shown to be required for Notch1 ubiquitination and down-regulation of Notch1 nuclear activity (56). A recent study has shown that Numb interacts with collapsing mediator protein-2 through the PTB domain, resulting in the regulation of Numb-mediated endocytosis at the growth cone; moreover, both Numb and collapsing mediator protein-2 have been shown to co-localize in the central region of axonal growth cones in hippocampal neurons (57). It has already been demonstrated that the interaction of 14-3-3 protein with its target proteins results in the regulation of subcellular localization, catalytic activity, or protein-protein interaction of the target proteins (52–55). Therefore, the phosphorylation-dependent interaction with 14-3-3 protein may affect the function of the PTB domain of Numb family proteins. Also, the CaM-KK/CaM-KI cascade has been shown to regulate growth cone motility (7). Thus, this kinase cascade may be integrated into the Numb-mediated pathways involved in axonal growth. Since the physiological consequences of the phosphorylation-dependent interaction of Numb/Numbl with 14-3-3 proteins remain to be examined, future experiments specifically designed to explore this issue will be necessary in order to better understand the regulatory mechanisms of the Numb family proteins.


   FOOTNOTES
 
* This work was supported in part by Grant-in aid for Scientific Research 17570115 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB210107 [GenBank] (rat Numbl) and AB210108 [GenBank] (rat Numb). Back

2 Present address: Dept. of Pharmacology, Emory University School of Medicine, Rollins Research Center, Room 5172, 1510 Clifton Rd., Atlanta, GA 30322. Back

1 To whom correspondence should be addressed: Dept. of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Tel./Fax: 81-87-891-2368; E-mail: tokumit{at}med.kagawa-u.ac.jp.

3 The abbreviations used are: CaM-K, Ca2+/CaM-dependent protein kinase; CaM-KK, CaM-K kinase; CaM, calmodulin; GST, glutathione S-transferase; DTT, dithiothreitol; CD, catalytic domain; CREB, cAMP-response element-binding protein; PKA, cAMP-dependent protein kinase, MS, mass spectrometry; LC, liquid chromatography; MS/MS, tandem mass spectrometry; PTB, phosphotyrosine binding. Back


   ACKNOWLEDGMENTS
 
We thank N. Ishikawa and T. Fujimoto (Kagawa University) for excellent technical assistance. We also thank Dr. Y. Watanabe (Kagawa University) for helpful discussion.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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