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J. Biol. Chem., Vol. 279, Issue 23, 24444-24451, June 4, 2004
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¶
From the
Department of Systems Biology, Harvard Medical School and Department of Cancer Cell Biology, The Dana-Farber Cancer Institute, Boston, Massachusetts 02115 and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, February 24, 2004 , and in revised form, April 1, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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87% sequence identity with NDR1. NDR2 is expressed in most human tissues with the highest expression in the thymus. In contrast to NDR1, NDR2 is excluded from the nucleus and exhibits a punctate cytoplasmic distribution. The differential localization of NDR1 and NDR2 suggests that each kinase may serve distinct functions. Thus, to identify proteins that interact with NDR1 or NDR2, epitope-tagged kinases were immunoprecipitated from Jurkat T-cells. Two uncharacterized proteins that are homologous to the Saccharomyces cerevisiae kinase regulators Mob1 and Mob2 were identified. We demonstrate that NDR1 and NDR2 partially colocalize with human Mob2 in HeLa cells and confirm the NDR-Mob interactions in cell extracts. Interestingly, NDR1 and NDR2 form stable complexes with Mob2, and this association dramatically stimulates NDR1 and NDR2 catalytic activity. In summary, this work identifies a unique class of human kinase-activating subunits that may be functionally analagous to cyclins. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although its precise function(s) remain unclear, evidence suggests that NDR1 may be involved in cell proliferation and/or tumor progression. First, NDR1 is hyperactivated in several S100B-positive melanoma cell lines (5). Given that S100B is overexpressed in greater than 80% of metastatic melanomas (6), NDR1 activity may influence tumor metastatic potential. Second, microdissection and gene expression profiling reveal that NDR1 mRNA is up-regulated 1.9-fold in high versus low risk ductal carcinoma in situ (7). Despite these intriguing observations, NDR1 has no known substrates and has not been implicated in any signal transduction pathway.
Substrate identification has undoubtedly been hampered by the seemingly low enzymatic activity of NDR1. Indeed, NDR1 expressed in either Escherichia coli or COS-1 cells is unable to transphosphorylate "generic" kinase substrates such as histone H1, myelin basic protein (MBP), casein, or phosvitin (1). Nevertheless, purified human NDR1 possesses catalytic activity. By screening a small peptide library, Hemmings and colleagues (5) identify a short peptide (KKRNRRLSVA) that serves as an NDR1 substrate. Taken together, these data suggest that either NDR1 has a very stringent substrate specificity or we have insufficient biological insight to purify active preparations of NDR1.
Mammalian genomes encode a related kinase, NDR2 (GenBankTM accession number NP_055815
[GenBank]
), which is 87% identical to NDR1 at the amino acid level (4, 8). Currently, the NDR2 kinase remains uncharacterized. However, a recent retroviral insertional mutagenesis study found that the murine NDR2 gene (also known as serine-threonine kinase 38-like) was disrupted in two independent B-cell lymphomas (9). This result suggests that NDR2 may also be involved in tumor initiation or progression. Given their potential involvement in cancer, we sought to characterize the human NDR1 and NDR2 kinases. In this study, we identified and characterized two new NDR1 and NDR2 binding partners, Mob1B and Mob2.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmids—The full-length NDR1 and NDR2 open reading frames (ORFs) were amplified by PCR from cDNA prepared from total HeLa RNA. The NDR1 ORF was cloned into the pECMV-FLAG (10) or pEGFP-C1 vector (Clontech) to generate pFLAG-NDR1 and pGFP-NDR1, respectively. Similarly, the NDR2 ORF was cloned into the pECMV-FLAG or pEGFP-C1 vector to generate pFLAG-NDR2 and pGFP-NDR2. Kinase-dead (KD) derivatives were generated by PCR using the QuikChange mutagenesis kit (Stratagene) with oligonucleotides that change the invariant Lys to Ala (K118A for NDR1, K119A for NDR2). Retroviral vectors were also constructed by cloning FLAG-NDR1 or FLAG-NDR2 ORFs into the murine stem cell virus (MSCV) vector pMSCV-puro (Clontech), generating pMSCV-FLAG-NDR1 and pMSCV-FLAG-NDR2. Retroviruses were packaged as described previously (11). Full-length Mob1B and Mob2 ORFs were amplified by PCR from cDNA prepared from Jurkat total RNA and inserted into pEGFP-C1 to generate pGFP-Mob1B and pGFP-Mob2, respectively.
Northern and Western Blot Analyses—To generate unique NDR1 and NDR2 probes, a 350-bp region at the 3' end of each cDNA was gel-purified and labeled with a Rediprime II labeling system (Amersham Biosciences). Radiolabeled probes were hybridized to a First-ChoiceTM Northern Human Blot I membrane (Ambion) in UltraHyb (Ambion), washed to a stringency of 0.2x SSC, 0.1% SDS at 42 °C, and exposed to a PhosphorImager (Amersham Biosciences). Western blot analysis was performed as described previously (11) using monoclonal anti-FLAG M2 (Sigma), monoclonal anti-NDR1 (BD Transduction Laboratories), monoclonal anti-p75/LEDGF (BD Transduction Laboratories), or polyclonal anti-GFP. BenchMarkTM protein ladder (Invitrogen) was used as a molecular weight standard for SDS-PAGE and Western blot analysis.
Identification of NDR1- and NDR2-interacting Proteins—Jurkat T-cells were infected with MSCV, MSCV-FLAG-NDR1, or MSCV-FLAG-NDR2 and expanded in the presence of 1 µg/ml puromycin. Whole cell extracts were prepared in immunoprecipitation buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 0.5% Triton X-100, 5% glycerol, 20 mM B-glycerophosphate, 1 mM sodium orthovanadate containing Complete protease inhibitors (Roche Applied Science)) and clarified by ultracentrifugation (20,000 rpm for 1.5 h at 4 °C using a Ti-70 rotor). Lysates were immunoprecipitated with anti-FLAG M2 affinity matrix (Sigma), washed extensively, and eluted with the FLAG peptide.
Immunoprecipitated protein were resolved by 4-20% Tris-glycine Novex SDS-PAGE and stained with Simply Blue (Invitrogen). The gel-resolved proteins were digested with trypsin, the mixtures were fractionated on a Poros 50 R2 RP microtip, and the resulting peptide pools were analyzed by matrix-assisted laser desorption ionization reflectron time-of-flight (MALDI-reTOF) mass spectrometry (MS) using a BRUKER UltraFlex TOF/TOF instrument (Bruker Daltonics) as described previously (12, 13). Selected experimental masses (m/z) were taken to search a nonredundant protein data base (1.4 x 106 entries, National Center for Biotechnology Information) utilizing the PeptideSearch (Matthias Mann, Southern Denmark University) algorithm with a mass accuracy restriction better than 40 ppm and no more than one missed cleavage site allowed per peptide. Mass spectrometric sequencing of selected peptides was done by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples using the UltraFlex instrument in the "LIFT" mode. Fragment ion spectra were taken to search nonredundant using the MASCOT MS/MS ion search program (Matrix Science Ltd.). Thus, any identification obtained was verified by comparing the computer-generated fragment ion series of the predicted tryptic peptide with the experimental MS/MS data. Hsp90
/
(GenBankTM accession numbers P07900
[GenBank]
and NP_031381
[GenBank]
, respectively) were identified as the 95-kDa band coimmunoprecipitating with FLAG-NDR1. Mob1B (GenBankTM accession number NP_775739
[GenBank]
; currently annotated as Mob4A yet referred to as Mob1B by Stavridi et al. (14)) was identified as the 26-kDa protein coimmunoprecipitating with FLAG-NDR2. Mob2 (GenBankTM accession number NP_443731
[GenBank]
) was identified as the 30-kDa protein coimmunoprecipitating with both FLAG-NDR1 and FLAG-NDR2. Of note, these tryptic peptides also matched to an unknown protein product of 16 kDa (BAB71443
[GenBank]
, but the mobility of the immunoprecipitated protein was much more consistent with Mob2 (27 kDa). A sequence analysis indicates that BAB71443
[GenBank]
is an alternately spliced variant of Mob2 (data not shown).
NDR1 and NDR2 Kinase Reactions—293T cells were transiently transfected with either pFLAG-NDR1 or pFLAG-NDR2 and pEGFP-C1, pGFP-Mob1B, or pGFP-Mob2. When indicated, the transfected cells were pulsed with 1 µM okadaic acid (Calbiochem) for 1 h prior to harvesting. At 45-h post-transfection, cells were washed with cold phosphate-buffered saline and then lysed in buffer containing 50 mM HEPES, pH 7.9, 1% Triton X-100, 250 mM NaCl, 50 mM NaF, 25 mM B-glycerophosphate, 1 mM sodium orthovanadate, and Complete protease inhibitors. Lysates were clarified by ultracentrifugation. Prior to immunoprecipitation, salt content was normalized to 500 mM NaCl. FLAG-NDR1 or FLAG-NDR2 complexes were immunoprecipitated with anti-FLAG M2 affinity resin, washed extensively in the above lysis buffer containing 500 mM NaCl and 0.1% Triton X-100, and then eluted in 50 mM HEPES, pH 7.9, 20% glycerol, 0.05% Triton X-100, 100 mM NaCl, 50 mM NaF, 25 mM B-glycerophosphate, 1 mM sodium orthovanadate, Complete protease inhibitors, and 200 µg/ml FLAG peptide (Sigma) and stored at -80 °C. In vitro kinase reactions were performed by incubating enzyme preparation in 25 mM HEPES, pH 7.9, 10 mM MgCl2, 1 mM dithiothreitol, and 100 µM [
-32P]ATP (1 µCi/µl) for 1 h at 30 °C. Transphosphorylation assays included 10 µg of dephosphorylated MBP (Upstate Biotechnology) and were performed for 20 min at 30 °C. Reactions were stopped by the addition of cold 2x SDS sample buffer. Samples were resolved by 12% SDS-PAGE, stained with Simply Blue, and exposed to a PhosphorImager.
Protein Alignments—The ClustalW algorithm was used to align the indicated protein sequences (15). Alignments were formatted with the ESPript tool (16).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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87% identical at the amino acid level (Fig. 1A). Most of the nonidentical residues cluster at the N and C termini of the enzymes, and these differences are highly conserved in the murine NDR1 and NDR2 sequences. Residues known to regulate NDR1 activity including Thr-74, Ser-281, and Thr-444 (reviewed in Ref. 8) are conserved in NDR2 and correspond to NDR2 residues Thr-75, Ser-282, and Thr-442. The NDR1 S100B-binding domain (residues 62-86) (5) contains one conservative and one nonconservative change in NDR2 (residues 63-87), whereas the NDR1 nuclear localization signal (residues 265-276) (1) contains a single conservative change in NDR2 (residues 266-277).
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Mob Proteins Coimmunoprecipitate with NDR1 and NDR2— To identify proteins that interact with NDR1 and NDR2, we generated Jurkat T-cell lines stably expressing FLAG-NDR1 or FLAG-NDR2. Following affinity purification, eluted samples were resolved by SDS-PAGE (Fig. 2A). Specific bands were excised from the gel, and the indicated proteins were identified by a combination of peptide mass fingerprinting using MALDI-TOF MS and mass spectrometric sequencing using MALDI-TOF/TOF MS/MS. We identified the 95-kDa band detected in NDR1 immunoprecipitations as Hsp90 (GenBankTM accession number P07900
[GenBank]
) and Hsp90 (GenBankTM accession number NP_031381
[GenBank]
). However, these proteins were also identified in Mock samples, suggesting that Hsp90/s partially adhere nonspecifically to the anti-FLAG resin under these conditions.
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/, we identified the 30-kDa band coimmunoprecipitating with both NDR1 and NDR2 as Mob2 (Fig. 2A) and the 26-kDa band coimmunoprecipitating with NDR2 as Mob1B. Of note, the 26-kDa band is not visible on the scanned Simply Blue-stained gel (top panel) but was readily observable following silver staining (bottom panel). A sequence analysis demonstrated that the identified proteins are homologous to the S. cerevisiae Mob1 and Mob2 proteins (Fig. 2B).
Mob2 Colocalizes with NDR1 and NDR2—We first sought to characterize the localization of Mob1B, the 26-kDa protein that was faintly detected in NDR2 immunoprecipitations (Fig. 2A). To this end, the full-length Mob1B ORF was cloned into an expression vector in-frame with an N-terminal GFP tag. GFP-Mob1B localization was then examined by fluorescence microscopy in transiently transfected HeLa cells. Mob1B exhibited both nuclear and diffuse cytoplasmic distribution when expressed alone (Fig. 3A). Coexpression of NDR1 (Fig. 3, B-D) or NDR2 (Fig. 3, E-G) did not noticeably alter the distribution of Mob1B. Consistent with our earlier observations (Fig. 1C), NDR1 localized predominantly to the nucleus with less prominent cytoplasmic speckles (Fig. 3C). NDR2 exhibited heterogeneous cellular distribution with predominantly cytoplasmic speckles (Fig. 3F). Although merged images suggest that Mob1B and NDR1 (Fig. 3D) or NDR2 (Fig. 3G) may have partially colocalized, interpretation of these results is difficult given the pan-cellular distribution of Mob1B.
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30-kDa protein that coimmunoprecipitated with both NDR1 and NDR2 (Fig. 2A). When expressed alone, Mob2 accumulated within the nucleus but was also observed within the cytoplasm (Fig. 3H). Coexpression of Mob2 and NDR1 (Fig. 3, I-K) or NDR2 (Fig. 3, L-N) dramatically altered Mob2 distribution. Under these conditions, Mob2 was mostly cytoplasmic with strong perinuclear staining (Fig. 3, I and L). Likewise, NDR1 exhibited reduced nuclear accumulation and more prominent cytoplasmic speckles (Fig. 3J), whereas NDR2 remained mostly cytoplasmic (Fig. 3M). The merged image demonstrates that NDR1 and Mob2 colocalized within the cytoplasm, particularly within perinuclear regions (Fig. 3K). Likewise, NDR2 and Mob2 colocalized predominantly in perinuclear regions but also throughout the cytoplasm (Fig. 3N). Thus, coexpression of NDR1 and Mob2 results in NDR1 redistribution from the nucleus (Figs. 1C and 3C) to the cytoplasm (Fig. 3J). Furthermore, NDR1 or NDR2 coexpression results in Mob2 relocalization to perinuclear regions (Fig. 3, compare I and L with H). Differential Binding of NDR Kinases to Mob1B and Mob2— The above analysis demonstrates that NDR1 and NDR2 colocalize with Mob2 in human cell lines. To extend these findings, we purified FLAG-NDR1 or FLAG-NDR2 from 293T cells following cotransfection with plasmids encoding GFP, GFP-Mob1B, or GFP-Mob2. Whole cell extracts were analyzed by anti-FLAG (Fig. 4A) and anti-GFP (Fig. 4B) Western blot to ensure equivalent expression of the respective FLAG and GFP fusion proteins. Following anti-FLAG affinity purification, a significant amount of Mob2 was coeluted with NDR1 and NDR2 (Fig. 4D). Mob1B also was coeluted with NDR2 (Fig. 4D) and, to a lesser extent, with NDR1 (see longer exposure in Fig. 4E). These results confirm that NDR1 and NDR2 bind both Mob1B and Mob2. Interestingly, the kinases appear to bind Mob2 more tightly than Mob1B in cell extracts, a finding consistent with our microscopy studies (Fig. 3). We also observed that the Mob1B-NDR1 interaction is significantly weaker than the Mob1B-NDR2 interaction, a finding consistent with our initial immunoprecipitations (Fig. 2A).
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In vitro kinase reactions were performed next using the above enzyme preparations and dephosphorylated MBP as substrate. NDR2 readily phosphorylated the 18-kDa MBP as well as the 24-kDa inactivated 
-phosphatase present within the MBP preparation (Fig. 5B). Two additional low abundance contaminants were phosphorylated by NDR2. These contaminants originated from the MBP preparation, because they were not observed in reactions in which MBP was omitted (data not shown). NDR2 autophosphorylation and Mob2 transphosphorylation were also observed (Fig. 5B), consistent with our previous findings (Fig. 4F). Reactions containing KD NDR2 exhibited negligible kinase activity, demonstrating that the NDR2 preparations were relatively pure.
NDR1 also phosphorylated MBP, albeit to a lesser extent than NDR2 (Fig. 5B). Interestingly, reactions containing KD NDR1 exhibited significantly more kinase activity than those containing KD NDR2. Given that significantly more Hsp90 copurified with KD NDR1 (Fig. 5B), the background enzymatic activity was probably the result of unknown kinase(s) associated with a subpopulation of Hsp90. Importantly, MBP transphosphorylation was invariably higher following incubation with WT NDR1 than with KD NDR1 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In contrast to NDR1, which is predominantly nuclear, NDR2 is localized predominantly to the cytoplasm. This observation is intriguing given the extremely high degree of sequence identity between the two enzymes. The punctate cytoplasmic distribution of FLAG-NDR2 and GFP-NDR2 (and to a lesser extent FLAG- and GFP-NDR1) suggests that a population of NDR2 may be restricted to a specific subcellular compartment.
Mob1B and Mob2 were identified as NDR1 and NDR2 binding partners. Transient transfection studies demonstrated that a fraction of Mob1B copurifies with NDR1 or NDR2 following immunoprecipitation under stringent conditions (500 mM NaCl, 1% Triton X-100). Mob2 tightly associates with NDR1 and NDR2 and is affinity-purified in a 1:1 stoichiometry with the kinases. At present, it is unclear whether the NDR kinases genuinely have a higher affinity for Mob2 than Mob1B. Indeed, it is possible that the interaction between NDR1 or NDR2 and Mob2 is salt- and/or detergent-sensitive or that the GFP moiety interferes with the interaction. We also demonstrated that NDR1 and NDR2 partially colocalize with Mob2 in human cell lines with the caveat that this analysis was performed with heterologously expressed proteins. Interestingly, the coexpression of Mob2 with NDR1 or NDR2 affects the subcellular localization of both proteins, and colocalization is mostly observed in regions surrounding the nucleus.
In vitro kinase reactions demonstrated that Mob2 dramatically stimulates the kinase activity of NDR1 and NDR2. We have shown that NDR1/2-Mob2 complexes are capable of both autophosphorylation and transphosphorylation of heterologous substrates. Interestingly, NDR2 is significantly more active than NDR1. At present, it is unclear whether NDR kinase activity is governed solely by primary sequence determinants or whether differential post-translational modifications account for increased NDR2 activity. In this respect, it is intriguing that NDR2 invariably migrates more slowly than NDR1 following SDS-PAGE despite the fact that the calculated molecular mass of NDR1 exceeds that of NDR2 by 187 Da.
Recently, Wu and colleagues (17) performed differential display PCR to identify genes preferentially expressed in human hepatocellular carcinomas (17). The authors cloned a 2520-bp mRNA that they named HCCA2 (hepatocellular carcinoma-associated gene 2) (GenBankTM accession number AF206328
[GenBank]
) and determined that HCCA2 mRNA was widely expressed in adult human tissues. Importantly, HCCA2 mRNA was detected in 79% (34/43) of primary hepatocellular carcinoma tissue samples but not in normal surrounding tissues. Furthermore, the expression of HCCA2 mRNA was correlated with Ki-67 protein expression and with tumor capsule invasion. The authors identified an ORF encoding a protein of 50 kDa within the HCCA2 mRNA (HCCA2 protein, GenBankTM accession number AAL74055
[GenBank]
and developed rabbit polyclonal antiserum against the C-terminal 160 amino acids of HCCA2 fused to GST. This antiserum detected a 50-kDa protein in 293 extracts transiently transfected with a pcDNA.3 vector containing the full-length HCCA2 cDNA but not in untransfected cell lysates. Western blot analysis of other samples either was not performed or was not reported. Interestingly, the HCCA2 mRNA also encodes an overlapping (but entirely distinct) ORF that was not described by Wu and colleagues (17). This alternate ORF encodes the Mob2 protein described here (GenBankTM accession number NP_443741
[GenBank]
). In our opinion, it is currently unclear whether the HCCA2 mRNA encodes one (Mob2) or both (Mob2 and HCCA2) proteins. In light of our analysis, it is tempting to reinterpret this report as evidence that Mob2 expression and thus NDR activation are associated with hepatocellular carcinoma development or progression.
The work presented here provides the first documentation of a serine-threonine kinase stimulated by a human Mob homologue. The proteins identified here are homologous to the S. cerevisiae Mob1 and Mob2 proteins (scMob1, scMob2). scMob1 and scMob2 were initially identified via a yeast two-hybrid interaction with the Mps1 serine-threonine kinase (hence, the name Mob, Mps One Binder) (18). MOB1 is an essential budding yeast gene required for cytokinesis and mitotic exit (18, 19). Aside from Mps1, scMob1 also binds the Dbf2 serine-threonine kinase (20) and regulates Dbf2 catalytic activity (3). scMob2 also forms a complex with the NDR family member Cbk1 and is important for maintaining polarized growth and regulation of the daughter cell-specific transcription factor, AceII (21). Interestingly, Schizosaccharomyces pombe Mob1 and Mob2 have been shown to interact with the fission yeast NDR kinase family members Sid2 and Orb6, respectively (22, 23). Taken together, our results show that Mob regulation of NDR activity has been conserved from yeast to humans.
It was previously hypothesized that scMob1 may activate Dbf2 by inducing a conformational switch analagous to cyclinmediated activation of Cdks (3). Recently, the crystal structure of human Mob1A (BAB14525
[GenBank]
was solved (Protein Data Base accession number 1PI1
[PDB]
) (14). Of note, Mob1A is 95% identical to Mob1B described here. The Mob1A structure revealed a negatively charged flat surface that is conserved among all of the Mob proteins that probably mediates electrostatic interactions with kinase binding partners (14). Although the structure of Mob1A does not resemble that of cyclins, cocrystal studies of Mob-NDR complexes might lend support to the cyclin-Cdk analogy. Regardless of the biophysical mechanism of activation, Mob proteins are conserved kinase-activating subunits. A sequence analysis demonstrated that the human genome contains seven Mob genes (data not shown). It will be interesting to determine whether other Mob proteins also regulate human NDR1, NDR2, or other members of the NDR kinase family. Given that NDR1, NDR2, and Mob2 have been implicated in melanomas, aggressive ductal carcinomas in situ, B-cell lymphomas, and hepatocellular carcinomas, molecular insight into the functions of the NDR kinases may lead to new therapeutic opportunities for the treatment of human cancers.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 617-632-5102; Fax: 617-632-5103; E-mail: pamela_silver{at}dfci.harvard.edu.
1 The abbreviations used are: NDR, nuclear Dbf2-related; MBP, myelin basic protein; ORF, open reading frame; KD, kinase-dead; GFP, green fluorescent protein; MALDI-reTOF, matrix-assisted laser desorption ionization reflectron time-of-flight; MS, mass spectrometry; HCCA2, hepatocellular carcinoma-associated gene 2; WT, wild-type; sc, S. cerevisiae; MSCV, murine stem cell virus; LEDGF, lens epithelialderived growth factor.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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