JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M409873200 on October 6, 2004

J. Biol. Chem., Vol. 279, Issue 50, 52191-52199, December 10, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/50/52191    most recent
M409873200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Davare, M. A.
Right arrow Articles by Soderling, T. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Davare, M. A.
Right arrow Articles by Soderling, T. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Inhibition of Calcium/Calmodulin-dependent Protein Kinase Kinase by Protein 14-3-3*

Monika A. Davare, Takeo Saneyoshi, Eric S. Guire, Sean C. Nygaard, and Thomas R. Soderling{ddagger}

From the Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239

Received for publication, August 27, 2004 , and in revised form, October 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular calcium concentrations regulate diverse cellular events including cytoskeletal dynamics, gene transcription, and synaptic plasticity. The calcium signal is transduced in part by the calcium/calmodulin-dependent protein kinase (CaMK) cascade that is comprised of CaMK kinase (CaMKK) and its primary downstream substrates, CaMKI and CaMKIV. The CaMK cascade also participates in cross-talk with other signaling pathways: CaMKK/CaMKI can activate the mitogen-activated protein kinase pathway and cAMP-dependent protein kinase (PKA) can directly phosphorylate two inhibitory sites (Thr108 and Ser458) in CaMKK. Here we report an additional PKA-dependent regulation of CaMKK through its interaction with protein 14-3-3. CaMKK and 14-3-3 co-immunoprecipitated from co-transfected heterologous cells as well as from rat brain homogenate, and site-directed mutagenesis studies identified phospho-Ser74 in CaMKK as the primary 14-3-3 binding site. In cultured rat hippocampal neurons and acute hippocampal slices this interaction was robustly stimulated by activation of PKA through forskolin treatment and was blocked by inhibition of PKA. Interaction of 14-3-3 with CaMKK had two regulatory consequences in vitro. It directly inhibited CaMKK activity, and it also blocked dephosphorylation of Thr108, an inhibitory PKA phosphorylation site. In human embryonic kidney 293 cells transfected with CaMKK and stimulated with forskolin, co-transfection with 14-3-3 prevented dephosphorylation of Thr108 to the same extent as did inhibition of protein phosphatases with okadaic acid. We conclude that binding of 14-3-3 to CaMKK stabilizes its inhibition by PKA-mediated phosphorylation, which may have important consequences in the regulation of CaMKI, CaMKIV, protein kinase B, and ERK signaling pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Ca2+/calmodulin-dependent protein kinase (CaMK)1 cascade is comprised of CaMKI, CaMKIV, and their upstream activator CaMKK (1, 2). Both CaMKI and CaMKIV have "activation loop" phosphorylation sites that, in the presence of elevated intracellular Ca2+, are phosphorylated by CaMKK, resulting in 10–20-fold increases in their activities (3, 4). CaMKI is present in most cell types where it is predominantly cytosolic (5), and recent studies implicate roles for it in regulation of mRNA translation (6), cytoskeleton organization (7), and axonal growth cone motility (8). CaMKIV has limited tissue distribution and is largely localized to the nucleus where it regulates gene transcription through phosphorylation of transcription factors such as CREB (9, 10) and its co-activator CBP (11). CaMKK can also slowly phosphorylate and activate PKB/Akt, and this appears to mediate in part the antiapoptotic effects of modest intracellular Ca2+ elevations (12). Thus, the CaMK cascade is integrally involved in major cellular functions.

As is true for most signal transduction systems, there is cross-talk between the CaMK cascade and other signaling pathways. For example, the cAMP-dependent protein kinase (PKA) can phosphorylate multiple sites in CaMKK (13), two of which are inhibitory (14, 15). Phosphorylation of Ser458, which is just C-terminal of the CaM-binding domain, partially suppresses activation of CaMKK by Ca2+/CaM. Alternatively binding of Ca2+/CaM to CaMKK blocks phosphorylation of Ser458 (16), so the regulation of CaMKK through this mechanism depends on the temporal sequence of signaling events. The CaMK cascade is also involved in Ca2+-dependent activation of ERK and c-Jun N-terminal kinase, members of the mitogen-activated protein kinase pathway. Depolarization of neuroblastoma NG108 cells produces strong and prolonged ERK activation that is blocked by the CaMKK inhibitor STO-609 (17). Dominant-negative constructs of nuclear localized CaMKIV or PKB/Akt had no effect on ERK activation, but dominant-negative CaMKI obviated Ca2+-dependent ERK activation. Furthermore depolarization stimulated neurite outgrowth, and this was also suppressed by inhibition of either CaMKK (STO-609) or ERK (U0126) (17).

Because CaMKK contains additional phosphorylation sites of unknown function (13), we performed a data base search (scansite.mit.edu), which revealed several predicted regulatory motifs including one for interaction with protein 14-3-3. The 14-3-3 family of acidic proteins contains seven isoforms that exist as dimers that bind to Ser/Thr-phosphorylated proteins and modulate their physiological properties (18–22). The prototypic (mode 1) 14-3-3 binding site has a Pro at the +2 position and an Arg at the –3 position relative to the phosphorylated Ser or Thr (RXXp(S/T)XP) (20). It should be noted that the –3 Arg is also a strong determinant for a number of protein kinases (e.g. PKA and CaMKII) (23–25). Some of the many phosphoproteins that are regulated by 14-3-3 interaction include Raf-1 (mitogen-activated protein kinase pathway) (20), BAD (proapoptotic protein) (26), Cbl (signaling adaptor protein) (27), Cdc25 (protein phosphatase) (28), and protein kinase C (29). Interaction of 14-3-3 with target phosphoproteins can modulate different functions including retention in the cytosol (Cdc25) (30), disruption of protein-protein interactions (BAD) (26), or inhibition of activity (protein kinase C) (29). Given the diverse roles of CaMKK in Ca2+ signaling, it was important to determine whether its functionality is regulated by 14-3-3. Here we report that PKA-mediated phosphorylation of Ser74 in CaMKK promotes its interaction with 14-3-3, resulting in direct inhibition of CaMKK activity as well as suppression of Thr108 dephosphorylation, thereby maintaining CaMKK in an inhibited state.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Monoclonal pan-14-3-3 antibody was purchased from Chemicon (Temecula, CA). Rabbit polyclonal 14-3-3{gamma} (C-16) and pan-14-3-3 (FL-246 and K-19) antibodies used for immunoprecipitation were from Santa Cruz Biotechnology (Santa Cruz, CA). The PKA site phospho-Ser/Thr-specific antibody was from Cell Signaling Technology (Beverly, MA). Monoclonal CaMKK{alpha}/{beta} antibody was from BD Biosciences Pharmingen. Forskolin, cyclosporine, and okadaic acid were from Alexis Biochemicals (San Diego, CA). H89 was purchased from Calbiochem. Recombinant PP2A and PP1 were purchased from Upstate Biotechnology (Waltham, MA) and New England Biolabs (Beverly, MA), respectively. Anti-phospho-CaMKI antibody has been described previously (17). 9E10 anti-Myc antibody was purified from hybridoma supernatant. SF9 purified recombinant PP2B was a gift Dr. Brian Perrino (University of Nevada, Reno, NV). Recombinant PKA was purified as described previously (14).

Constructs—CaMKK{alpha} was subcloned by PCR into pGEX-4T3, and truncation constructs of CaMKK{alpha} were generated by PCR. Site-directed mutagenesis was performed following the manufacturer's protocol (Stratagene) for generation of glutathione S-transferase (GST)-CaMKK mutants (S74A, T108G, S458A, S475A, and T108G/S458A/S475A). Mammalian Myc-tagged 14-3-3 isoforms were a gift of Dr. Alaistair Aitken (National Institute for Medical Research, London, UK). For bacterial expression, the 14-3-3 isoforms were subcloned using PCR into pGEX-4T3 (GST fusion protein) and pRSET-A (His6 fusion proteins). Mammalian expression vector for CaMKK{alpha} was generated by amplifying the coding region of rat CaMKK{alpha} isoform by PCR from pME18SCaMKK{alpha} wild type by using the following primers: rCamKK U EcoRI (5'-ggaattcggagcgcagtccagccg-3') and rCamKK D Asp718I (5'-ccggtacctcaggatgcagcctcatcttc-3'). The PCR product was digested with EcoRI-Asp718I, purified by gel electrophoresis, and subcloned into the ClaI-Asp718I site of expression plasmid pCAGGS (31) with ClaI-EcoRI fragment from pCSMT (32) to generate pCAGGS Myc-CaMKK{alpha}.

GST Fusion Protein Expression, Pull-down Assays, and in Vitro Binding Assays—GST-CaMKK{alpha}, GST-14-3-3, His6-14-3-3{gamma}, and {tau} constructs were expressed and purified as described previously (33). For pull-down assays, 1–4 µg of GST fusion protein was loaded on glutathione-Sepharose. When indicated, GST-CaMKK{alpha} was phosphorylated in vitro with PKA (1 µg/ml) and ATP for 20 min at 32 °C. The PKA phosphorylation reaction was stopped by addition of 2 µM PKA inhibitor peptide (PKI) and washing with 4-fold excess volume of lysis buffer. For pull-down experiments, rat brain cytosol (with addition of 0.5% Triton X-100) was added to the immobilized GST-CaMKK and incubated with rocking at 4 °C for 2–4 h. To remove unbound proteins, the beads were washed two to three times with lysis buffer followed by SDS-PAGE. Bound 14-3-3 was detected by immunoblotting with pan-14-3-3 antibodies.

Cell Culture and Transfection—HEK293 and COS7 cells were cultured as described previously (14). For transfection experiments, ~4 x 105 cells were plated in each well of a 6-well plate 24 h prior to transfection. Cells were transfected with 1.5 µg of total plasmid DNA/well using FuGENE reagent according to the manufacturer's protocol (Roche Applied Science). 24 to 36 h post-transfection, cells were washed once in ACSF (HEPES-based) and incubated with the indicated pharmacological inhibitors for 30–60 min. Stimulations were done as described under "Results." Cells were instantly frozen in liquid N2 and stored at –80 °C until further processing or lysed immediately for immunoprecipitations.

Acute Hippocampal Slice Preparation—Adult male Sprague-Dawley rats (6–8 weeks old) were anesthetized with pentobarbital (60 mg/kg intraperitoneally) and sacrificed by decapitation. The brains were removed within 1 min of decapitation and immediately submerged in ice-cold, oxygenated (95% O2, 5% CO2) sucrose-ACSF for hippocampal dissection: 110 mM sucrose, 60 mM NaCl, 2.5 mM KCl, 28 mM NaHCO3, 1.25 mM NaH3PO4, 0.5 mM CaCl2, 7 mM MgCl2, 5 mM glucose, 0.6 mM sodium ascorbate, pH 7.4 at 4 °C. Hippocampal slices (400 µm, transverse) were prepared in ice-cold sucrose-ACSF using a Vibratome and transferred as cut to warm, oxygenated ACSF (125 mM NaCl, 2.5 mM KCl, 21.4 mM NaHCO3, 1.25 mM NaH3PO4, 2 mM CaCl2, 1 mM MgCl2, 11.1 mM glucose, pH 7.4 at 32 °C) for recovery (30 min at 37 °C and then the chamber was equilibrated for 1.5–2 h at 22 °C). Slices from the dorsal and ventral thirds of the hippocampus were discarded. Following recovery, slices were transferred to ACSF at 32 °C and equilibrated for 1 h, pretreated with H89, 11-arginine PKI peptide, or 11-arginine control peptide and then treated with or without 25 µM forskolin for 5 min. Slices were kept submerged at all times. Poststimulation the slices were immediately lysed by homogenization in 1% Triton X-100 in a glass Dounce homogenizer. The lysates were cleared by ultracentrifugation (100,000 x g, 30 min), and cleared lysates were used for immunoprecipitation as described.

Immunoprecipitation and Immunoblotting—For immunoprecipitation, rat forebrain, acute hippocampal slices, hippocampal cultures, or transfected heterologous cells were lysed in lysis buffer (1% Triton X-100, 137 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM EGTA, pH 7.4, 5 mM EDTA, pH7.4, 20 mM NaF, 20 mM NaPPi, 1 mM {beta}-glycerol phosphate, protease inhibitors (leupeptin, aprotinin, antipain, phenylmethylsulfonyl fluoride, and pepstatin A), and 1 µM microcystin. Insoluble material was cleared by ultracentrifugation (hippocampal slices and rat forebrain: 50,000 rpm for 30 min) or centrifugation at 14,000 rpm for 20 min in a table top, refrigerated Eppendorf centrifuge (cultured cells). 4–10 µg of indicated antibody was added to about 500–700 µg of cleared lysate and incubated either for 1 h or overnight on ice. Immobilized protein A (Repligen Inc.) or protein G-Sepharose (Amersham Biosciences) was added, and lysates were further incubated for 1–2 h while tilting at 4 °C. Immunoprecipitated complexes were washed twice with a 4-fold excess volume of lysis buffer and once with 10 mM HEPES alone. Samples were then extracted, and proteins were detected by SDS-PAGE and immunoblotting. Initial immunoblotting (Figs. 1, 2, 3, 4) was done with blocking, primary, and secondary antibodies diluted in 5% milk followed by horseradish peroxidase-based signal detection with ECL Plus (Amersham Biosciences) and exposure to x-ray film (Biomax MR and ML from Eastman Kodak Co.). Subsequent (Figs. 5, 6, 7) immunoblotting was performed in accordance with the Odyssey near infrared imaging platform system (LI-COR), and data were captured using the Odyssey scanner. For quantification of data, the x-ray film-based Western blot data were scanned and densitized using Kodak imaging software, and the data gathered using the Odyssey system were densitized with the same system software. All the data were further processed and normalized using Microsoft Excel, and statistical analyses were performed using Prism software.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1.
CaMKK binds protein 14-3-3. Glutathione-Sepharose beads containing either GST alone or GST-CaMKK{alpha} were subjected to either mock, PKA, or autophosphorylation (Ca2+/CaM) conditions. The immobilized proteins were incubated with rat brain cytosol as a source of protein 14-3-3 and then washed extensively. Bound 14-3-3 was detected by 10% SDS-PAGE and immunoblotting with pan-14-3-3 antibody (upper blot). The lower blot shows loading of GST-CaMKK (93 kDa) or GST (28 kDa) detected by Ponceau S staining.

 



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 2.
CaMKK association with 14-3-3 requires PKA phosphorylation of Ser74. A, schematic diagram of CaMKK constructs illustrating the catalytic domain (CATALYTIC, residues 126–434), regulatory domain (REG, residues 435–463) containing overlapping autoinhibitory and CaM-binding motifs (16), and site-specific mutations. B, immobilized wild-type (wt) and mutant GST-CaMKK constructs, without or with PKA phosphorylation, were incubated with rat brain cytosol as a source of 14-3-3. Bound 14-3-3 was detected by 10% SDS-PAGE and immunoblotting with pan-14-3-3 antibody. Fusion protein loading is demonstrated with Ponceau S staining (lower panel).

 



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 3.
14-3-3 isoform selectivity of CaMKK interaction. A, COS7 cells were transiently transfected with Myc-tagged 14-3-3 {epsilon}, {eta}, {gamma}, and {zeta} isoforms or mock-transfected. Cell supernatants were applied to PKA-phosphorylated GST-CaMKK-immobilized beads for 3 h at 4 °C followed by washing. Bound 14-3-3 was detected by immunoblotting with anti-Myc antibody (left panel), and 3% of the lysate was blotted to determine expression levels (right panel). B, GST fusions of the indicated 14-3-3 isoforms were used in pull-down assays with a 1% Triton X-100 rat brain extract as the source of CaMKK.

 



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 4.
Co-immunoprecipitation of CaMKK and 14-3-3. A, left panel, 14-3-3 (lane 1) or non-immune rabbit IgG (lane 2) were used for immunoprecipitation from 1% Triton X-100-solubilized rat forebrain extract. 2% of the input was used as a loading control (lane 3). Right panel, exogenous cAMP (1 mM), ATP (0.4 mM), or PKI (1 µM) was added to the brain homogenate (50 mM HEPES, 10 mM MgCl2) and incubated at 32 °C for 20 min. H.C., heavy chain of IgG. B, acute hippocampal slices were submerged and recovered in ACSF and preincubated with phosphate-buffered saline, 1 µM 11-arginine PKI peptide (PKI-11R) (PKA inhibitor), 10 µM H89, or 1 µM 11-arginine control peptide (Ctrl 11R) in ACSF for 1 h at 32 °C. Slices were then stimulated as indicated with 25 µM forskolin (F), dideoxyforskolin (dideF) (inactive analog), or Me2SO (vehicle (Veh)) for 10 min at 37 °C. Hippocampal slices were immediately lysed, and immunoprecipitations were done as in A. A representative blot is shown in the left panel, and quantification of multiple experiments is shown in the right panel (n = 6 (vehicle and forskolin), n = 3 (H89 and 11-arginine PKI), and n = 2 (11-arginine control peptide). *, p ≤ 0.05; ***, p ≤ 0.005. Rb., rabbit; IP, immunoprecipitation.

 



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 5.
Interaction of 14-3-3 with CaMKK inhibits catalytic activity. Wild-type (A) or T108G/S458A mutant (B) CaMKK{alpha} bound to glutathione-Sepharose beads was phosphorylated with PKA or mock-phosphorylated (no ATP added) at 32 °C for 20 min. Phosphorylation reactions were stopped by addition of the PKA inhibitor peptide PKI and by washing with ice-cold radioimmune precipitation assay buffer. Beads were incubated with bovine serum albumin (–14-3-3), 14-3-3{gamma}, or 14-3-3{tau} as indicated for 1 h at 4 °C. After washing, catalytically inactive CaMKI was added as substrate, and the CaMKK bound to beads was assayed (1.5 min at 30 °C) for its ability to phosphorylate the activation loop (Thr177) in CaMKI. The phosphorylation was detected using an anti-phospho-Thr177 CaMKI antibody (8, 17). The left panels demonstrate representative Western blots for pCaMKI (upper panel), total CaMKI (middle panel), and total CaMKK (bottom panel) in the final reaction mixtures. The right panels show quantification of four to six independent experiments. **, p ≤ 0.01; ***, p ≤ 0.005.

 



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 6.
14-3-3 prevents dephosphorylation of Thr108 by protein phosphatase 2A. A, GST-CaMKK fusion proteins (wild type and indicated mutants) were phosphorylated in vitro with PKA, and phosphorylation was detected using an anti-phospho-PKA site (Ser/Thr) antibody (Cell Signaling Technology). Note that the antibody detects only Thr108 phosphorylation in CaMKK (left panel, upper blot). Blots were reprobed with anti-GST for protein loading (left panel, lower blot). Phosphorylation (normalized to the protein amount) is shown in the right panel. B, GST-CaMKK constructs (wild type (wt) or S74A) were subjected to phosphorylation without or with PKA, the reactions were stopped with PKI, and His-tagged 14-3-3{gamma}, GST-His6, or phosphate-buffered saline was added at room temperature for 15 min. The CaMKK mixture was added to a protein phosphatase reaction buffer with or without PP2A (0.1 unit), and dephosphorylation was carried out at 30 °C for 20 min. The phosphorylation status of Thr108 in CaMKK was detected by immunoblotting with anti-phospho-PKA Ser/Thr site antibody (upper blots), which only detects Thr108 phosphorylation (see A), and blots were Western blotted for CaMKK. Representative blots from one experiment are shown. C, quantification of three independent experiments. In each set of results, the +PKA and –phosphatase result was set at 100%, and the rest of the data were normalized to this. **, p ≤ 0.01.

 



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 7.
Expression of 14-3-3{gamma} inhibits CaMKK dephosphorylation at Thr108 in HEK293 cells. HEK293 cells were co-transfected with Myc-CaMKK and either pCDNA3.1 or FLAG-14-3-3{gamma}. At 24 h post-transfection, cells were washed once with ACSF and incubated for 25–40 min with Me2SO (vehicle) or protein phosphatase inhibitors, okadaic acid (OA, 0.2 µM), or cyclosporine A (CsA, 50 µM) followed by stimulation for 5 min with forskolin (Fsk, 25 µM) to activate PKA or with Me2SO (vehicle). Cells were lysed in radioimmune precipitation assay buffer, and CaMKK was immunoprecipitated with anti-Myc antibody. Thr108 phosphorylation was detected by Western blotting with phospho-PKA Ser/Thr site antibody as in Fig. 6. A representative blot is shown in A, and quantification from three to five independent experiments is depicted in B. **, p ≤ 0.01; ***, p ≤ 0.005.

 
Kinase Activity Assay—Immobilized GST-CaMKK{alpha} was incubated with kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 0.4 mM ATP, and protease inhibitors) and phosphate-buffered saline for mock phosphorylation or with PKA (1 µg/0.5 ml) at 32 °C for 10–20 min. The reaction was stopped with the addition of 1 µM PKI. The samples were then either extracted with SDS sample buffer and processed for SDS-PAGE or used for the phosphatase assay.

Phosphatase Assay—For PP1 and PP2A, reactions were done in a buffer consisting of 50 mM HEPES, pH 7.4, 5 mM MgCl2, 5 mM MnCl2, 0.5 µM dithiothreitol, and protease inhibitors for the indicated times at 32 °C. Phosphatase activities were matched using p-nitrophenyl phosphate hydrolysis experiments prior to dephosphorylation experiments. For PP2B, 2 mM CaCl2, 2 µM calmodulin, and 8 mM sodium ascorbate was added to the above phosphatase buffer. PP2B activities were typically 5-fold lower than PP1 and PP2A even though the amount of protein used was comparable or greater for PP2B.

Quantification and Statistics—Analysis of variance with Tukey's post-test was used to test whether statistical significance existed among treated samples. To determine significance for two treatments compared with each other, a paired two-tail t test was performed with significance threshold set at p ≤ 0.05. Where indicated in the figures, a single asterisk denotes p ≤ 0.05, a double asterisk denotes p ≤ 0.01, and a triple asterisk indicates p ≤ 0.005.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PKA Phosphorylation of CaMKK Regulates Interaction with Protein 14-3-3—In addition to PKA-mediated phosphorylation of Thr108 and Ser458 that directly inhibits CaMKK activity (14, 15), PKA also phosphorylates Ser52, Ser74, and Ser475 with unknown regulatory consequences (13). Ser475 was identified by a web site search as a putative protein 14-3-3 interaction site (ERSMS475APGN). The Arg at position 472 (–3 position relative to Ser475) and the Pro at position 477 (+2 position) are positive determinants for 14-3-3 interaction (34). We initially tested for interaction between CaMKK and 14-3-3 with a pull-down assay using recombinant GST-tagged CaMKK{alpha} as bait and rat brain cytosol as a source for native 14-3-3. Immobilized GST-CaMKK{alpha} was used without treatment (Fig. 1, lanes 1 and 2) or with mock phosphorylation with PKA (absence of ATP) (lanes 3 and 4), PKA phosphorylation (lanes 5 and 6), or autophosphorylation in the presence of calcium, calmodulin, and ATP (lanes 7 and 8) and subsequently incubated with rat forebrain cytosol as a source of native 14-3-3. Immobilized GST alone was used as a control in the pull-down assay (lanes 9 and 10). Immunoblotting with pan-14-3-3 antibodies revealed specific binding of rat brain 14-3-3 to PKA-phosphorylated CaMKK{alpha} (lanes 5 and 6). The lower panel (Fig. 1) of Ponceau S staining shows equivalent protein loading for the GST-CaMKK{alpha} or the GST alone. These data demonstrate for the first time a specific interaction between CaMKK{alpha} and 14-3-3 that is dependent on phosphorylation of CaMKK by PKA.

Mapping the Primary 14-3-3 Interaction Site to Ser74 of CaMKK{alpha}—Although Ser475 was previously identified as a PKA site in CaMKK, its phosphorylation level was low and only occurred in the presence of Ca2+/CaM (13). To determine whether Ser475 is the PKA phosphorylation site required for 14-3-3 binding, this site was mutated to Ala, and the GST pull-down experiment was repeated. Surprisingly these experiments showed no change in 14-3-3 binding comparing wild-type versus S475A CaMKK{alpha} (data not shown). Furthermore a synthetic peptide containing the sequence around Ser475 was not significantly phosphorylated by PKA (data not shown). Because previous work has shown that PKA phosphorylates CaMKK{alpha} on Thr108 and Ser458 (13–15), we tested whether either of these sites was responsible for binding of 14-3-3 by generating the triple mutant T108A/S458A/S475A (see schematic in Fig. 2A). However, this triple mutant construct of GST-CaMKK{alpha} still robustly interacted with 14-3-3 (Fig. 2B, middle panel). In fact, the triple mutant exhibited stronger interaction and pulled down additional 14-3-3 isoforms (lower band). Perhaps elimination of these PKA phosphorylation sites enhances the phosphorylation stoichiometry of the critical site(s) required for 14-3-3 binding (see below).

To identify the 14-3-3 interaction site in CaMKK, two GST constructs were made, one lacking the first 107 amino acids of CaMKK and the other containing only those N-terminal 107 residues (Fig. 2A, lower two constructs). Pull-down assays with these constructs demonstrated strong interaction of 14-3-3 with the N1–107 GST construct in a PKA phosphorylation-dependent manner (Fig. 2B, right panel, lanes 7 and 8). 14-3-3 binding was completely abrogated by deletion of the N terminus of CaMKK (Fig. 2B, right panel, lanes 5 and 6). These data clearly demonstrate that the 14-3-3 interaction site resides in the first 107 amino acids (N terminus) of CaMKK{alpha}, so we tested the GST-CaMKK{alpha} mutants S52A and S74A. The Ser52 mutant showed a slight reduction in PKA-dependent 14-3-3 binding (data not shown), but mutation of Ser74 eliminated interaction with 14-3-3 (Fig. 2B, right panel, lanes 3 and 4). The wild-type and N1–107 CaMKK{alpha} (positive controls) proteins confirmed that 14-3-3 was pulled down in the assay when Ser74 was present. We conclude that phospho-Ser74 in CaMKK{alpha} serves as the "gatekeeper" site for 14-3-3 interaction (35) even though it is not a "canonical" 14-3-3 sequence.

14-3-3 Isoform Selectivity for CaMKK Interaction—To further our understanding of the functional consequence of the CaMKK{alpha} interaction with 14-3-3, we investigated whether CaMKK{alpha} preferentially binds to any specific 14-3-3 isoform(s) (36). COS7 cells were mock- or transiently transfected with Myc epitope-tagged expression vectors of 14-3-3 isoforms. CaMKK{alpha} preferentially bound to 14-3-3 {eta} and {gamma} isoforms from these lysates (Fig. 3A, left panel, lanes 5–8), whereas the {epsilon} and {zeta} isoforms did not bind (Fig. 3A, left panel, lanes 1 and 2 and lanes 9 and 10, respectively). Similar Myc-14-3-3 expression levels were observed in the lysates (Fig. 3A, right panel).

To further examine this isoform selectivity, we expressed and purified recombinant GST-tagged 14-3-3 {epsilon}, {eta}, {gamma}, {tau}, and {zeta} proteins. The GST-14-3-3 proteins were immobilized on glutathione-Sepharose and used for pull-down assays with 1% Triton X-100 rat brain extract as the source of native CaMKK. Rat brain CaMKK was pulled down with highest efficiency by 14-3-3{gamma} (Fig. 3B, upper panel, lanes 5 and 6). The sequence surrounding Ser74, the interaction site on CaMKK, contains an arginine in the –3 position and a glutamine in the +2 position (RKFSLQ). Previous work has shown that the 14-3-3{gamma} isoform can interact with substrates containing glutamine in the +2 position (37), consistent with our data showing a preferential interaction of CaMKK with 14-3-3{gamma}.

CaMKK and 14-3-3 Co-precipitate from Brain—We demonstrated a strong interaction between CaMKK{alpha} and 14-3-3 in vitro using GST pull-down assays. However, for this interaction to have physiological relevance, we needed to confirm the existence of this protein complex in cells. When CaMKK{alpha} was transiently transfected in COS7 cells, we observed co-immunoprecipitation of endogenous 14-3-3 with transfected CaMKK (data not shown). Furthermore, when FLAG-tagged CaMKK{alpha} or CaMKK{beta} was expressed in HEK293 cells and affinity purified, interaction with 14-3-3 was detected by mass spectroscopy2 as described previously (38).

Because CaMKK and 14-3-3{gamma} are highly expressed in brain, we tested whether endogenous CaMKK{alpha} interacts with native 14-3-3 in rat forebrain extracts. CaMKK co-immunoprecipitated with 14-3-3{gamma} from adult rat brain homogenate but not with non-immune rabbit IgG (Fig. 4A, left panel, lanes 1 and 2). To determine whether the interaction of CaMKK with 14-3-3 can be modulated by activation of PKA, brain homogenates were incubated with exogenous cAMP plus ATP without or with a PKI (39) to modulate phosphorylation of native CaMKK. CaMKK co-immunoprecipitated as before under control conditions, and this association was stimulated by addition of cAMP/ATP (Fig. 4A, right panel, upper blot, lanes 1 and 2) and was blocked by addition of PKI peptide to the homogenate (Fig. 4A, right panel, upper blot, lanes 3).

Using acute rat hippocampal slices, we next tested whether CaMKK interaction with native 14-3-3 can be stimulated in intact neurons. Acute slices were preincubated at 32 °C with vehicle (Me2SO) or the PKA inhibitors H89 and membrane-permeable 11-arginine PKI or a scrambled control 11-arginine peptide for 1 h. Slices were then stimulated with 20 µM forskolin to generate cAMP and activate PKA for 5 min at 32 °C in ACSF. A 14-3-3{gamma}-specific antibody was used for immunoprecipitation from cleared tissue lysates. CaMKK co-immunoprecipitation with 14-3-3 from slices was enhanced about 7-fold by forskolin treatment but not with vehicle or dideoxyforskolin (Fig. 4B, left panel, upper blot, lanes 1, 2, and 4). This forskolin-dependent stimulation of CaMKK binding to 14-3-3 was dependent on PKA since the PKA inhibitors H89 and membrane-permeable 11-arginine PKI blocked this interaction, whereas the control 11-arginine peptide did not (Fig. 4B, right lanes). These data, summarized in the graph of Fig. 4B, clearly demonstrate a strong stimulation of 14-3-3 binding to CaMKK{alpha} upon activation of PKA in intact cells. These experiments using hippocampal slices were reproduced in cultured (16–21 days in vitro) hippocampal neurons (data not shown).

14-3-3 Interaction Regulates Catalytic Activity of CaMKK— Having established that CaMKK interacts with protein 14-3-3 in neurons, it was important to determine what functions of CaMKK might be regulated by 14-3-3. Interactions of other proteins with 14-3-3 have been shown to regulate their subcellular localization (cytoplasmic versus nuclear partition) or catalytic function or perturb protein-protein interactions (19, 35, 40). Thus, several possibilities exist for the functional regulation of CaMKK by 14-3-3. Although the primary motif for 14-3-3 interaction with CaMKK, Ser74, is somewhat N-terminal to the catalytic domain of CaMKK{alpha} (Fig. 2A), we examined whether 14-3-3 binding might affect the catalytic activity of CaMKK{alpha} using a pull-down kinase assay. GST-CaMKK was immobilized on glutathione-Sepharose without or with PKA phosphorylation. The GST-CaMKK was then incubated without or with His6-14-3-3{gamma} or His6-14-3-3{tau}. Since 14-3-3{tau} did not interact with CaMKK{alpha} in previous experiments, this served as a control for 14-3-3{gamma}. The complex of 14-3-3 bound to GST-CaMKK was washed to remove excess, unbound 14-3-3. This complex of CaMKK without or with bound 14-3-3 was used to test the activity of CaMKK{alpha}. A well established downstream target of CaMKK is Thr177 in the activation loop of CaMKI (3). We used purified recombinant, catalytically inactive (to prevent autophosphorylation) CaMKI as a substrate for testing the activity of CaMKK, and phosphorylation of Thr177 was detected using a phosphospecific antibody for this site (8, 17). CaMKK strongly phosphorylated CaMKI (Fig. 5A, left panel, upper blot, lanes 1 and 2), and this was not altered by addition of 14-3-3{gamma} in the absence of PKA phosphorylation (lanes 3–5), indicating that there were no nonspecific effects of 14-3-3 on the kinase assay. When wild-type CaMKK was phosphorylated by PKA, we observed a strong inhibition of CaMKK catalytic activity in the absence of 14-3-3{gamma} (p ≤ 0.001) (Fig. 5A, left panel, upper blot, lanes 6–8, and bar graph). This inhibitory effect of PKA phosphorylation on CaMKK has been described previously and is a result of phosphorylation of Thr108 and Ser458 (14, 15). When 14-3-3{gamma} binds PKA-phosphorylated CaMKK, we observed an additional small but significant inhibition of catalytic activity (p ≤ 0.01) (Fig. 5A, left panel, upper blot, lanes 9–11, and bar graph). This inhibition of CaMKK activity is specific for binding of 14-3-3{gamma} to CaMKK since the addition 14-3-3{tau}, an isoform that does not bind CaMKK, did not show a significant further decrease in catalytic activity. The levels of CaMKI phosphorylation were reflective of CaMKK catalytic activity since all assays had equivalent amounts of CaMKI and CaMKK (Fig. 5A, left panel, middle and lower blots, respectively). All the immunoblot data were densitized and normalized as (phospho-CaMKI/total CaMKI)/total CaMKK. The mean of the normalized data for CaMKK activity in the absence of PKA phosphorylation and 14-3-3 was given a relative value of 100, and the rest of the data set were normalized to this. These data suggest that interaction of 14-3-3 with phospho-CaMKK can further depress its activity.

Because PKA phosphorylation of Thr108 and Ser458 strongly suppresses CaMKK activity, it was difficult to examine the inhibitory effect of 14-3-3{gamma} binding to phopho-Ser74. To obviate this problem, we used the double mutant T108G/S458A. With this CaMKK mutant, PKA cannot phosphorylate and inhibit CaMKK via Thr108 and Ser458 but will still phosphorylate Ser74 and result in 14-3-3 binding (Fig. 2B). This should allow us to specifically test the inhibitory effect of 14-3-3 interaction with CaMKK as these mutations have little effect on the activity of CaMKK{alpha}. As expected, PKA phosphorylation of this mutant CaMKK had no affect on its catalytic activity (Fig. 5B, left panel, upper blot, lanes 6–8, and bar graph). However, addition of 14-3-3{gamma}, but not 14-3-3{tau}, to this PKA-phosphorylated CaMKK mutant resulted in about 60% inhibition of CaMKK catalytic activity (Fig. 5B, left panel, upper blot, lanes 9–11). These data with the T108G/S458A CaMKK{alpha} construct demonstrate that interaction with 14-3-3{gamma} has the potential to significantly inhibit CaMKK catalytic activity.

14-3-3 Blocks Dephosphorylation of Thr108 by Protein Phosphatase 2A—Since interaction of 14-3-3 with Ser74, either through steric or conformational effects, can alter the activity of CaMKK, we tested whether this interaction might also regulate dephosphorylation of CaMKK. We determined that a commercial "anti-PKA" site phosphospecific antibody detects PKA phosphorylation of CaMKK only at Thr108. As shown in Fig. 6A, this phosphospecific antibody recognizes PKA-phosphorylated wild-type CaMKK as well as the mutants S74A and S458A but not T108G. Thus, we used this anti-PKA phospho-Ser/Thr antibody to specifically assess levels of Thr108 phosphorylation in CaMKK and whether interaction of 14-3-3 blocks dephosphorylation of this inhibitory site by protein phosphatases.

Initial studies showed that PP2B exhibited poor dephosphorylation of Thr108 under our experimental conditions (data not shown), so it was not further examined. Because 14-3-3 has been reported to interact with the catalytic subunits of both PP1 and PP2A (22), we tested whether 14-3-3 directly inhibited either phosphatase using p-nitrophenyl phosphate as substrate. The presence of 14-3-3{gamma} (0.2 µg/µl) gave slight (10–15%) inhibition of both phosphatases in this assay (data not shown). With PKA-phosphorylated CaMKK as substrate, PP2A gave robust dephosphorylation of Thr108, and this dephosphorylation was completely blocked by the presence of 14-3-3 (Fig. 6B). To confirm that the protection from dephosphorylation of Thr108 by PP2A was indeed mediated by 14-3-3{gamma} binding to Ser74, we repeated the same experiment except for the use of the S74A mutant that cannot bind 14-3-3. With the S74A mutant, 14-3-3 had no effect on dephosphorylation of Thr108, confirming that the effect of 14-3-3 was due to its binding to CaMKK. Although PP1 also gave strong dephosphorylation of Thr108 in CaMKK, the suppression of dephosphorylation by 14-3-3 was observed with both wild-type and the S74A mutant (data not shown), making interpretation of the results difficult. These data indicate that 14-3-3{gamma} plays a prominent role in inhibiting CaMKK activity both by active inhibition of catalysis and also by prolonged maintenance of phosphorylation of the neighboring inhibitory site, Thr108.

Protection by 14-3-3{gamma} of Phospho-Thr108 by 14-3-3 from Dephosphorylation in HEK293 Cells—To investigate whether 14-3-3 protects against dephosphorylation of phospho-Thr108 CaMKK in intact cells, we transiently co-transfected HEK293 cells with Myc-tagged CaMKK plus either pcDNA3.1 (empty vector) or FLAG-tagged 14-3-3{gamma}. 24–36 h post-transfection the cells were preincubated with either vehicle (Me2SO) or the phosphatase inhibitors okadaic acid or cyclosporine A for 30 min at 37 °C and then treated with 20 µM forskolin for 10 min to stimulate PKA-mediated phosphorylation of CaMKK. Transfected CaMKK was immunoprecipitated using anti-Myc antibody, and the phosphorylation state of Thr108 was determined by Western blotting with anti-PKA phospho-Ser/Thr antibody (described above). In the absence of transfected 14-3-3{gamma}, forskolin produced a 4–5-fold increase in phosphorylation of Thr108. Treatment by cyclosporine A, a selective and potent membrane-permeable inhibitor of PP2B (41), had no effect on the phosphorylation status of Thr108 (Fig. 7A, left panel, lane 3), suggesting that PP2B is relatively inactive toward Thr108 under these conditions. Okadaic acid (42), a potent inhibitor of PP2A that can also block PP1, resulted in a dramatic increase in forskolin-stimulated phosphorylation of Thr108 (Fig. 7A, left blots, lanes 4) due to inhibition of PP2A and perhaps PP1.

We showed in vitro that interaction of 14-3-3{gamma} prevents dephosphorylation of Thr108 (Fig. 6B). If this regulation occurs in cells, co-transfection with 14-3-3{gamma} should mimic the effect of okadaic acid to enhance Thr108 phosphorylation. In congruence with this hypothesis, co-transfection with 14-3-3{gamma} increased Thr108 phosphorylation after forskolin stimulation to a level equivalent to that observed by okadaic acid treatment (Fig. 7A, right panel, lanes 1–4). These data demonstrate that 14-3-3{gamma} regulates the phosphorylation state of Thr108 in intact cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Elevated intracellular calcium triggers multiple signaling pathways including the CaMK cascade, consisting of CaMKK and its downstream targets CaMKI, CaMKIV, and PKB that regulate diverse physiological responses such as neuronal growth cone motility (8), gene transcription (9–11), and apoptosis (12). Here we demonstrate a new cellular mechanism for regulation of CaMKK activity through its interaction with protein 14-3-3. Binding of 14-3-3 to CaMKK requires its prior phosphorylation of Ser74 by PKA. Thus, when PKA is activated in the cell, it phosphorylates at least three regulatory sites in CaMKK, Ser74, Ser108, and Ser458. The latter two phosphorylation sites contribute to inhibition of CaMKK activity (14, 15), whereas the major consequence of Ser74 phosphorylation is to allow binding of 14-3-3 that can also directly inhibit CaMKK as well as prevent subsequent dephosphorylation of Ser108, thereby prolonging inhibition of CaMKK.

Specificity of 14-3-3 Interaction with CaMKK—The 14-3-3 proteins are ubiquitously expressed phosphoserine/phosphothreonine-binding proteins that are members of a large family of isoforms (18–22). As expected, binding of 14-3-3 to CaMKK required phosphorylation by PKA, but it was surprising that Ser475, the predicted interaction site in CaMKK that contains an Arg in the –3 position and a proline at +2, was not responsible. Although Ser475 is a predicted consensus PKA site, it is poorly phosphorylated and only in the presence of Ca2+/CaM binding to CaMKK (13). Although Ser74 has an arginine in the –3 position, there is a glutamine rather than the prototypic proline in the +2 position (RKFS74LQ). Ligands with glutamine in the +2 position have been shown to selectively interact with the 14-3-3{gamma} isoform (37). Consistent with this selectivity, CaMKK appears to preferentially bind the {gamma} isoform of 14-3-3 in vitro. Furthermore, when an antibody specific for the 14-3-3{gamma} isoform was used for immunoprecipitation from rat brain extract, CaMKK was co-precipitated, confirming that CaMKK does form a complex with 14-3-3{gamma} in brain. We do not understand why CaMKK interacted equally with both the {gamma} and {eta} isoforms of 14-3-3 in Fig. 3A but predominantly with the {gamma} isoform in Fig. 3B. Perhaps the rat brain CaMKK in Fig. 3B had other modifications that reduced its binding to the {eta} isoform. Regulation of CaMKK by 14-3-3 may be selective for cell types that are enriched in both proteins. We chose hippocampal neurons for our studies because their cell bodies are highly enriched in CaMKK (1, 2) as well as the {gamma} isoform of 14-3-3 (43). Although our studies focused on the {alpha} isoform of CaMKK, it appears that 14-3-3 also interacts with the {beta} isoform of CaMKK since they co-immunoprecipitated when overexpressed in COS or HEK293 cells (data not shown).

Dynamic Regulation of CaMKK and 14-3-3 Interaction in Brain—If the interaction of CaMKK with 14-3-3 is physiologically relevant, then modulation of the phosphorylation state of Ser74 should dictate this interaction. Treatment of hippocampal slices with forskolin, a potent adenylyl cyclase activator, increased this interaction 7-fold as determined by co-immunoprecipitation. As predicted, inhibition of PKA with either H89 or the specific PKA inhibitor peptide PKI attenuated binding of 14-3-3 to CaMKK. While it is clear that PKA can regulate this interaction through phosphorylation of Ser74, it will be interesting to determine whether other kinases might also phosphorylate Ser74 since the –3 arginine is a positive determinant for several protein kinases. If other kinases phosphorylate Ser74 but not Thr108 or Ser458, this would elicit the direct inhibition of CaMKK by 14-3-3.

Functional Consequences of the Interaction of CaMKK with 14-3-3—As mentioned in the Introduction, interaction of 14-3-3 with its various target proteins can have multiple functional consequences. The 14-3-3 dimer is a rigid structure that can act as an "allosteric clamp" to modify enzyme conformation and activity (44) in response to a signaling pathway that phosphorylates the gatekeeper site. According to this hypothesis there is a primary gatekeeper phosphorylation site (e.g. Ser74) necessary for initial binding of one of the 14-3-3 subunits. Once bound, the second subunit of the 14-3-3 dimer can interact with a secondary site in the target protein, thereby producing an allosteric clamp in the target protein. This secondary site can be another phospho-Ser/Thr (26).

In the specific case of CaMKK, binding of 14-3-3 inhibits kinase activity through two mechanisms. The first is direct inhibition of CaMKK activity. This is most clearly seen using the T108G/S458A mutant that was not directly inhibited by PKA phosphorylation of these two sites. With this mutant, binding of 14-3-3 resulted in ~60% inhibition. Several examples have been reported where binding of 14-3-3 directly inhibits catalytic activity (45–49). However, it appears that the major effect of 14-3-3 binding to PKA-phosphorylated CaMKK may be to block subsequent dephosphorylation of Thr108. Thus, interaction of 14-3-3 with CaMKK maintains the kinase in an inhibited state. This conclusion was confirmed in HEK293 cells since transfection with 14-3-3{gamma} blocked dephosphorylation of Thr108 as effectively as did inhibition of protein phosphatases with okadaic acid. In the presence of 14-3-3, forskolin produced a 10-fold stronger phosphorylation of Thr108, indicating that protein phosphatases rapidly dephosphorylate this inhibitory site in CaMKK in the absence of bound 14-3-3. Unfortunately we do not have a phosphospecific antibody to monitor the phosphorylation state of the other inhibitory site, Ser458.

Physiological Role of CaMKK Inhibition in Relation to Other Signaling Pathways—It is interesting to speculate why it may be physiologically important to suppress CaMKK activity when PKA is activated. Since parallel pathways are capable of stimulating multiple aspects of cellular function, suppression of one pathway while the other is active would produce signaling specificity. Thus, CaMKK activity appears to be subject to complex combinatorial regulation depending on the activity state of PKA and/or other kinases that phosphorylate Ser74, 14-3-3{gamma} isoform expression, or subcellular localization. For example, both PKA and CaMKIV, a downstream target of CaMKK, impinge on CREB-mediated transcription in the nucleus. The role of CaMKIV in physiological regulation of CREB phosphorylation is not entirely clear, but it appears that CaMKIV catalyzes acute but not sustained CREB phosphorylation upon depolarization of neurons (10). Perhaps more importantly, activated CaMKIV can regulate transcription through phosphorylation of Ser301 in the CREB co-activator CBP (11). This phosphorylation of CBP by CaMKIV regulates Ca2+-dependent CBP-mediated transcription. It will be interesting to see whether prior activation of PKA suppresses Ca2+-dependent CaMKIV-mediated phosphorylation of CBP and transcriptional readout.

A second physiological function that is enhanced by both CaMKK and PKA is axonal outgrowth in neurons. The CaMKK and CaMKI pathway maintains basal axonal extension through regulation of growth cone morphology and motility (8), whereas activation of PKA stimulates axonal extension through phosphorylation of synapsin 1 (50). Consequences of activation of both pathways in this developmental process have not been investigated. Third, both PKA and the CaMKK/CaMKI pathway modulate apoptosis. Depending on the cell type, PKA activation can either promote or suppress apoptosis. One apparent mechanism for protection against apoptosis is PKA-mediated phosphorylation of the proapoptotic factor BAD (51, 52). This phosphorylation promotes interaction of BAD with 14-3-3, thereby sequestering BAD. CaMKK can activate PKB/Akt (12), which also phosphorylates BAD and promotes its binding to 14-3-3 (53). Lastly PKA activation results in either activation or inhibition of ERK by the Rap1 GTPase depending on whether a given cell type expresses B-Raf (54). Elevation of intracellular Ca2+ through cell depolarization can mediate ERK activation through the CaMKK and CaMKI pathway (17). Thus, prior activation of PKA could suppress the ability of CaMKK/CaMKI to activate ERK through 14-3-3{gamma} binding to CaMKK. PKA can also block activation of ERK by phosphorylation of Raf-1 on serines 33, 233, and 259 followed by recruitment of 14-3-3 that inhibits kinase activity (48). Since CaMKK, through activation of CaMKI, can also activate ERK (17), 14-3-3 may exert multiple mechanisms to suppress ERK activation in response to cellular elevations of cAMP.

These signaling pathways are very complex as they are dictated in large part by developmental, spatial, and temporal parameters. There are discrete spatial patterns of 14-3-3{gamma} expression during development in brain (55). A significant portion of cellular PKA responses are restricted due to compartmentalization of PKA with A kinase anchoring proteins to various organelles (56). Furthermore prior binding of Ca2+/CaM to CaMKK can modify the ability of PKA to phosphorylate certain sites (13). For example, PKA does not phosphorylate Ser458, which is at the C terminus of the CaM-binding domain, when Ca2+/CaM is bound (16). Thus, one might predict that elevation of Ca2+ prior to PKA activation may blunt the inhibition of CaMKK by PKA. Therefore, it is difficult to predict the cellular consequences of inhibition of CaMKK by PKA, and future experiments specifically designed to explore these issues will be necessary to understand the physiological impact of this cross-talk.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 GM41292 (to T. R. S.) and NS27037 (to T. R. S.) and Training Grant DK007680 (to M. A. D.) and by Human Frontier Science Program Fellowship LT00193 (to T. S.). 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. Back

{ddagger} To whom correspondence should be addressed: Vollum Inst. L-474, Oregon Health and Sciences University, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-6931; Fax: 503-494-4534; E-mail: soderlit{at}ohsu.edu.

1 The abbreviations used are: CaMK, calcium/calmodulin-dependent protein kinase; CaMKK, CaMK kinase; CaM, calmodulin; PKA, cAMP-dependent protein kinase; HEK, human embryonic kidney; ERK, extracellular signal-regulated kinase; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; PKB, protein kinase B; PP, protein phosphatase; PKI, PKA inhibitor peptide; ACSF, artificial cerebrospinal fluid. Back

2 T. Saneyoshi and T. Natsume, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Alaistair Airken (National Institute for Medical Research, London, UK) for the 14-3-3 expression constructs and the Soderling laboratory for constructive comments and technical assistance during this project.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Soderling, T. R. (1999) Trends Biochem. 24, 232–235[CrossRef][Medline] [Order article via Infotrieve]
  2. Hook, S. S., and Means, A. R. (2001) Annu. Rev. Pharmacol. Toxicol. 41, 471–505[CrossRef][Medline] [Order article via Infotrieve]
  3. Haribabu, B., Hook, S. S., Selbert, M. A., Goldstein, E. G., Tomhave, E. D., Edelman, A. M., Snyderman, R., and Means, A. R. (1995) EMBO J. 14, 3679–3686[Medline] [Order article via Infotrieve]
  4. Selbert, M. A., Anderson, K. A., Huang, Q. H., Goldstein, E. G., Means, A. R., and Edelman, A. M. (1995) J. Biol. Chem. 270, 17616–17621[Abstract/Free Full Text]
  5. Picciotto, M. R., Zoli, M., Bertuzzi, G., and Nairn, A. C. (1995) Synapse 20, 75–84[CrossRef][Medline] [Order article via Infotrieve]
  6. Qin, H., Raught, B., Sonenberg, N., Goldstein, E. G., and Edelman, A. M. (2003) J. Biol. Chem. 278, 48570–48579[Abstract/Free Full Text]
  7. Suizu, F., Fukuta, Y., Ueda, K., Iwasaki, T., Tokumitsu, H., and Hosoya, H. (2002) Biochem. J. 367, 335–345[CrossRef][Medline] [Order article via Infotrieve]
  8. Wayman, G. A., Kaech, S., Grant, W. F., Davare, M., Impey, S., Tokumitsu, H., Nozaki, N., Banker, G., and Soderling, T. R. (2004) J. Neurosci. 24, 3786–3794[Abstract/Free Full Text]
  9. Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269, 15520–15527[Abstract/Free Full Text]
  10. Bito, H., Deisseroth, K., and Tsien, R. W. (1996) Cell 87, 1203–1214[CrossRef][Medline] [Order article via Infotrieve]
  11. Impey, S., Fong, A. L., Wang, Y., Obrietan, K., Wayman, G. A., Storm, D. R., Soderling, T. R., and Goodman, R. H. (2002) Neuron 34, 235–244[CrossRef][Medline] [Order article via Infotrieve]
  12. Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584–587[CrossRef][Medline] [Order article via Infotrieve]
  13. Okuno, S., Kitani, T., and Fujisawa, H. (2001) J. Biochem. (Tokyo) 130, 503–513[Abstract/Free Full Text]
  14. Wayman, G. A., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073–16076[Abstract/Free Full Text]
  15. Matsushita, M., and Nairn, A. C. (1999) J. Biol. Chem. 274, 10086–10093[Abstract/Free Full Text]
  16. Tokumitsu, H., Wayman, G. A., Muramatsu, M., and Soderling, T. R. (1997) Biochemistry 36, 12823–12827[CrossRef][Medline] [Order article via Infotrieve]
  17. Schmitt, J. M., Wayman, G. A., Nozaki, N., and Soderling, T. R. (2004) J. Biol. Chem. 279, 24064–24072[Abstract/Free Full Text]
  18. Yaffe, M. B., and Elia, A. E. (2001) Curr. Opin. Cell Biol. 13, 131–138[CrossRef][Medline] [Order article via Infotrieve]
  19. Fu, H., Subramanian, R. R., and Masters, S. C. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 617–647[CrossRef][Medline] [Order article via Infotrieve]
  20. Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. (1996) Cell 84, 889–897[CrossRef][Medline] [Order article via Infotrieve]
  21. Tzivion, G., and Avruch, J. (2002) J. Biol. Chem. 277, 3061–3064[Free Full Text]
  22. Rubio, M. P., Geraghty, K. M., Wong, B. H., Wood, N. T., Campbell, D. G., Morrice, N., and Mackintosh, C. (2004) Biochem. J. 379, 395–408[CrossRef][Medline] [Order article via Infotrieve]
  23. Pearson, R. B., Woodgett, J. R., Cohen, P., and Kemp, B. E. (1985) J. Biol. Chem. 260, 14471–14476[Abstract/Free Full Text]
  24. Payne, M. E., Schworer, C. M., and Soderling, T. R. (1983) J. Biol. Chem. 258, 2376–2382[Abstract/Free Full Text]
  25. Kemp, B. E., Bylund, D. B., Huang, T. S., and Krebs, E. G. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3448–3452[Abstract/Free Full Text]
  26. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996) Cell 87, 619–628[CrossRef][Medline] [Order article via Infotrieve]
  27. Liu, Y. C., Liu, Y., Elly, C., Yoshida, H., Lipkowitz, S., and Altman, A. (1997) J. Biol. Chem. 272, 9979–9985[Abstract/Free Full Text]
  28. Peng, C. Y., Graves, P. R., Thoma, R. S., Wu, Z., Shaw, A. S., and Piwnica-Worms, H. (1997) Science 277, 1501–1505[Abstract/Free Full Text]
  29. Hausser, A., Storz, P., Link, G., Stoll, H., Liu, Y. C., Altman, A., Pfizenmaier, K., and Johannes, F. J. (1999) J. Biol. Chem. 274, 9258–9264[Abstract/Free Full Text]
  30. Dalal, S. N., Schweitzer, C. M., Gan, J., and DeCaprio, J. A. (1999) Mol. Cell. Biol. 19, 4465–4479[Abstract/Free Full Text]
  31. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193–199[CrossRef][Medline] [Order article via Infotrieve]
  32. Turner, D. L., and Weintraub, H. (1994) Genes Dev. 8, 1434–1447[Abstract/Free Full Text]
  33. Atkins, C. M., Nozaki, N., Shigeri, Y., and Soderling, T. R. (2004) J. Neurosci. 24, 5193–5201[Abstract/Free Full Text]
  34. Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J., and Cantley, L. C. (1997) Cell 91, 961–971[CrossRef][Medline] [Order article via Infotrieve]
  35. Yaffe, M. B. (2002) FEBS Lett. 513, 53–57[CrossRef][Medline] [Order article via Infotrieve]
  36. Vincenz, C., and Dixit, V. M. (1996) J. Biol. Chem. 271, 20029–20034[Abstract/Free Full Text]
  37. Rittinger, K., Budman, J., Xu, J., Volinia, S., Cantley, L. C., Smerdon, S. J., Gamblin, S. J., and Yaffe, M. B. (1999) Mol. Cell 4, 153–166[CrossRef][Medline] [Order article via Infotrieve]
  38. Natsume, T., Yamauchi, Y., Nakayama, H., Shinkawa, T., Yanagida, M., Takahashi, N., and Isobe, T. (2002) Anal. Chem. 74, 4725–4733[Medline] [Order article via Infotrieve]
  39. Scott, J. D., Fischer, E. H., Demaille, J. G., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4379–4383[Abstract/Free Full Text]
  40. Takahashi, Y. (2003) Neurochem. Res. 28, 1265–1273[CrossRef][Medline] [Order article via Infotrieve]
  41. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Cell 66, 807–815[CrossRef][Medline] [Order article via Infotrieve]
  42. Haystead, T. A., Sim, A. T., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78–81[CrossRef][Medline] [Order article via Infotrieve]
  43. Baxter, H. C., Liu, W. G., Forster, J. L., Aitken, A., and Fraser, J. R. (2002) Neuroscience 109, 5–14