Cloning and Characterization of MST4, a Novel Ste20-like Kinase
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m009323200
ISSN1083-351X
AutoresZhijian Qian, Clark Lin, Rafael Espinosa, Michelle M. LeBeau, Marsha Rich Rosner,
Tópico(s)Cancer-related Molecular Pathways
ResumoMST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized. Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3. Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase. Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situhybridization. Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase. An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues. MST4a lacks an exon encoding kinase subdomains IX–XI that stabilizes substrate binding. The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase. MST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized. Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3. Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase. Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situhybridization. Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase. An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues. MST4a lacks an exon encoding kinase subdomains IX–XI that stabilizes substrate binding. The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase. mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase p21 Rac/Cdc42-activated kinase polymerase chain reaction myelin basic protein hemagglutinin Mitogen-activated protein kinases (MAPKs)1 are highly conserved mediators of signal transduction that are present in all eukaryotes and play essential roles in regulating cell differentiation, cell proliferation, and cell death. The core of the cascade consists of three sequentially acting protein kinases: a MAPK, a MAPK kinase, and a MAPK kinase kinase (1Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar). In yeast, there are five MAPK cascades that have pivotal roles in regulating sporulation, cell wall remodeling, osmolyte synthesis, filamentation, and mating (2Herskowitz I. 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EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). Thus, PAKs can act as upstream activators of the MAPK cascades in mammalian cells. The second subfamily of Ste20-like kinases, in which germinal center kinase is the prototype (19Katz P. Whalen G. Kehrl J.H. J. Biol. Chem. 1994; 269: 16802-16809Abstract Full Text PDF PubMed Google Scholar), is characterized by an N-terminal kinase domain followed by a C-terminal regulatory domain. This subfamily also includes HPK1 (20Hu M.C. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar, 21Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (200) Google Scholar), GLK (22Diener K. Wang X.S. Chen C. Meyer C.F. Keesler G. Zukowski M. Tan T.H. Yao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9687-9692Crossref PubMed Scopus (120) Google Scholar), NIK( (23Su Y.C. Han J. Xu S. 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Gene ( Amst. ). 1995; 167: 303-306Crossref PubMed Scopus (119) Google Scholar, 31Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and MST3 and MST3b (32Schinkmann K. Blenis J. J. Biol. Chem. 1997; 272: 28695-28703Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 33Zhou T.H. Ling K. Guo J. Zhou H. Wu Y.L. Jing Q. Ma L. Pei G. J. Biol. Chem. 2000; 275: 2513-2519Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Germinal center kinase, GLK, HGK, HPK, NIK, and KHS have been shown to activate the JNK pathway at the level of MAPK kinase kinase (21Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (200) Google Scholar, 22Diener K. Wang X.S. Chen C. Meyer C.F. Keesler G. Zukowski M. Tan T.H. Yao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9687-9692Crossref PubMed Scopus (120) Google Scholar, 23Su Y.C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (219) Google Scholar, 24Tung R.M. Blenis J. Oncogene. 1997; 14: 653-659Crossref PubMed Scopus (67) Google Scholar, 28Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (137) Google Scholar, 34Ling P. Yao Z. Meyer C.F. Wang X.S. Oehrl W. Feller S.M. Tan T.H. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar). MST1, MST2, MST3, MST3b, and SOK1 have extensive homology in their C-terminal tail but have not been demonstrated to directly activate any of the known MAPK cascades (28Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (137) Google Scholar). However, there are reports that MST1 can activate JNK and p38 (35Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (325) Google Scholar), and MST3 can potentiate ERK activation under certain conditions (33Zhou T.H. Ling K. Guo J. Zhou H. Wu Y.L. Jing Q. Ma L. Pei G. J. Biol. Chem. 2000; 275: 2513-2519Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In the present study, using a screen for Raf-interacting proteins, we cloned and characterized MST4, a new member of the mammalian Ste20 family that has a strong homology to mammalian Ste20-like kinases MST1, MST2, MST3, and SOK1. The existence of a new member of the MST family, as well as an alternatively spliced, inactive isoform, suggests that these enzymes may play discrete and specific roles in regulating MAPK signaling pathways. The catalytic domain of Raf was amplified by PCR with primer 1 (5′-gacggatccgttcacagccgaaaacccccgtgcc-3′) and primer 2 (5′-cgaccatggctagaagacaggcagcctcgg-3′), digested with XhoI and BamHI, and subcloned into PEG202 to construct PEG202-c-Raf. MEK1 was amplified by PCR with primer 3 (5′-gcctcgagcccaagaagaagccgacg-3′) and primer 4 (5′-gcctcgagtcagatgctggcagcgtg-3′), digested with XhoI, and subcloned into PJG4–5 to construct PJG-4–5-MEK. The PEG202-Raf was transformed into Epicurian coli XL1-red cells (Stratagene), which are deficient in three of the primary DNA repair pathways. After recovery in SOC medium (GIBLO BRL) at 37 °C for 1 h, the culture was diluted to 10 ml of SOC for further incubation overnight. The next day, 200 μl of the culture was diluted with a fresh 10 ml of SOC and grown overnight, while the remaining culture was used for plasmid DNA preparation. The second overnight culture was further diluted for another overnight incubation and plasmid DNA preparation. A third overnight culture was also used for preparation of plasmid DNA. These three plasmid DNA preparations were pooled together as the mutated PEG202-c-Raf library, which was transformed into yeast EGY48 along with PJG-MEK in order to screen for a mutated PEG202-Raf protein that has a high affinity for MEK. A mutant construct was isolated (PEG202-Raf-M), and we confirmed that the mutant Raf protein had an enhanced affinity for MEK by retransforming the PEG202-c-Raf-M construct back into yeast EGY48 with PJG4–5-MEK and testing its interaction with MEK. Sequencing the c-Raf insertion in PEG202 revealed a mutation at residue Ser610 that introduced a new stop codon at this site, resulting in a truncated C-terminal Raf protein. The LexA yeast two-hybrid system was kindly provided by Dr. Roger Brent. A human fetal library, made from a 22-week-old human fetal frontal cortex, was used to search for proteins interacting with the kinase domain of mutant PEG202-Raf-M (see above). Two clones showing strong interaction with Raf were obtained. Routine yeast work and yeast transformation were performed as described (36Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1324) Google Scholar). cDNA from two human fetal brain libraries, a human testes library, and multiple human tissue cDNA panels from CLONTECH were analyzed by polymerase chain reaction using primer MST-761 (5′-ctactaagattccgaatcagagccctc-3′) and primer MST-1 (5′-atggcccactcgccggtggctgtc-3′). A ∼1-kilobase pair DNA fragment was amplified by PCR, purified using a Qiagen PCR purification kit, and labeled with 32P using the Megaprime DNA labeling kit (Amersham Pharmacia Biotech). The probe was hybridized to a poly(A)+ RNA human multiple tissue Northern blot (CLONTECH) following the user manual. COS and 293 cells were grown in a 95% air, 5% CO2 incubator at 37 °C. Dulbecco's modified Eagle's medium supplemented with antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin) and 10% fetal bovine serum was used for cell growth. Transfection of COS and 293 cells was performed using the TransITTM polyamine transfection reagent (Panvera, Madison, WI). For lysis, cultured cells were washed twice with ice-cold phosphate-buffered saline and lysed in 1% Triton-based lysis buffer containing 1% Triton X-100, 100 mm NaCl, 50 mm Tris-HCl, pH 7.5, 50 mm NaF, 40 mm β-glycerophosphate, 2 mm EDTA, 1 mm sodium vanadate, 1 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 20 mm ρ-nitrophenyl phosphate. A primer (5′-gtcgttgctattcgaatcatagac-3′) was used for unique site elimination site-directed mutagenesis of MST4a to generate the kinase inactive mutant MST4a-K53R. The critical lysine in the kinase domain (VAIKIID) was mutated to arginine. The mutagenesis was performed according to the manual for the Amersham Pharmacia Biotech U.S.E. mutagenesis kit. The smaller BamHI/BsmI DNA fragment from PCR3.1-MST4a-K53R was ligated into the largerBamHI/BsmI DNA fragment from PCR3.1-MST4 to construct PCR3.1-MST4-K53R. Primer FLAG-MST-5′ (5′-gccgccatggactacaaggacgacgatgacaaggcccactcgccggtggctg-3′) and primer MST-3′-1165 (5′-ccatcgatcatggagctcatgggttaag-3′) were used to clone the MST4a from PJG-MST4 by PCR and clone MST4 from Marathon-readyTM cDNA by PCR with aCLONTECH PCR Advantage 2 kit. The PCR products were purified by the Qiagen PCR purification kit and subcloned into the TA cloning vector PCR3.1 (Invitrogen) to construct PCR3.1-MST4a and PCR3.1-MST4. Primer FLAG-MST-5′ and primer MST-761 (5′-ctactaagattccgaatcagagccctc-3′) were used to clone the MST4-NT from Placenta Marathon-ready cDNA by PCR. The PCR products were ligated into the TA cloning vector PCR3.1 (Invitrogen) to construct the N-terminal construct PCR3.1-MST4-NT. The FLAG-MST-5′ primer contained the FLAG epitope right after the ATG code and the 19 nucleotides in the 5′-end of MST4. The plasmids were sequenced by the interdisciplinary center for biotechnology research sequencing core laboratory at the University of Florida or by the University of Chicago Cancer Research Center DNA sequencing facility. For MST4, FLAG-MST4a, FLAG-MST4, FLAG-MST4-K53R, or FLAG-MST4-NT were transfected into cells, grown in regular medium for 24 h, and starved in serum-free medium or grown in regular medium for 48 h. Following lysis in 1% Triton X-100 buffer, the cell extracts were incubated with protein G beads coated with anti-FLAG M5 antibody for 3 h. The immune complex was washed three times with lysis buffer and two times with kinase buffer (20 mm Hepes, pH 7.4., 10 mmMgCl2, 1 mm MnCl2, 1 mmdithiothreitol, 0.2 mm sodium vanadate, 10 mmρ-nitrophenyl phosphate). 5 μg of MBP substrate was used per reaction in kinase buffer containing 5 μCi of [γ-32P]ATP. The kinase reaction was incubated for 25 min at 30 °C and then boiled with sample buffer for 5 min. The reaction products were separated on 12% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. Western blot analysis was performed as described previously (37Abe M.K. Chao T.-S., O. Solway J. Rosner M.R. Hershenson M.B. Am. J. Resp. Cell. Mol. Biol. 1994; 11: 577-585Crossref PubMed Scopus (75) Google Scholar). For MAPK assays, either HA-ERK2, HA-JNK, or HA-p38 was expressed in cells along with FLAG-MST4 expression vectors and/or Myc-Raf-1 expression vectors as indicated and immunoprecipitated with anti-HA antibodies as above. Kinase activities were monitored by immunoblotting with the appropriate anti-phospho-pTXpY antibodies (New England Biolabs, Boston). Human metaphase cells were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes. The MST4 probe was a cDNA probe containing full-length MST4a. Fluorescence in situ hybridization was performed as described previously (38Camoretti-Mercado B. Forsythe S.M. LeBeau M.M. Espinosa III, R. Vieira J.E. Halayko A.J. Willadsen S. Kurtz B. Ober C. Evans G.A. Thweatt R. Shapiro S. Niu Q. Qin Y. Padrid P.A. Solway J. Genomics. 1998; 49: 452-457Crossref PubMed Scopus (74) Google Scholar). Biotin-labeled probes were prepared by nick translation using Bio-16-dUTP (Enzo Diagnostics). Hybridization was detected with fluorescein-conjugated avidin (Vector Laboratories), and chromosomes were identified by staining with 4,6-diamidino-2-phenylindole dihydrochloride. A human fetal brain library was employed in a LexA-based yeast two-hybrid screen to identify proteins that interact with Raf. A modified Raf bait was created by mutagenizing the yeast plasmid PEG202 containing the catalytic domain of Raf and selecting for enhanced interaction with MEK1 (see "Materials and Methods"). Using the modified Raf bait plasmid, we obtained two cDNA clones that coded for the same previously uncharacterized cDNA. Sequence analysis showed that both clones encoded a polypeptide of 354 amino acids, which was later termed MST4a (Fig.1). Eukaryotic protein kinases have a common catalytic core structure in their kinase domain, which typically contains 11 conserved subdomains (39Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2296) Google Scholar). Comparison of the MST4a protein sequence with that of MST family members revealed that MST4a lacked part of the kinase domain. In order to see if MST4a might be a splicing variant of a full-length MST cDNA, the kinase subdomains I–XI in MST4 cDNAs from three separate libraries were examined by PCR analysis. As shown in Fig.2 (left panel), two PCR products with different sizes were amplified from a fetal human brain (CLONTECH) cDNA library that was different from the human brain cDNA library used in the original yeast two-hybrid screen as well as from a testes and a placenta library. Sequence analysis indicated that the smaller PCR product had the same sequence as the kinase domain of MST4a, and the larger PCR product had an additional cDNA insert. Subcloning the full-length fragments into an expression vector and subsequent sequencing revealed that the larger PCR product corresponded to the full-length MST cDNA containing the missing IX, X, and XI kinase domains (MST4, Fig. 1). The deduced protein sequence of MST4, consisting of 416 amino acids (Fig. 3 A), has a kinase domain at the N terminus and a regulatory domain at the C terminus. Comparison of the protein sequence of MST4 with other enzyme sequences (Fig. 3 B) indicated that the kinase domain is most closely related to the catalytic domains of Mammalian Ste20-like kinases MST1 (51% identity), MST2 (54% identity), MST3 (65% identity), and SOK1 (63% identity). The C-terminal regulatory domain of MST4 is most similar to the C-terminal domains of MST3 and SOK1.Figure 3Nucleotide and predicted amino acid sequence of human MST4 and sequence alignment between MST4 and other mammalian Ste20-like kinases. A, cDNA and predicted protein sequence of the full-length MST4. B, sequence comparison between MST4 and other members of the MST family. The predicted amino acid sequence of MST4 was compared with that of the mammalian Ste20-like kinases MST1, MST2, MST3, and SOK1 by Geneworks. Amino acids conserved in all protein are shaded.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Most of the genomic DNA sequence of human MST4 has been mapped. Interestingly, there is a putative 186-base pair exon corresponding to kinase subdomains IX–XI that, if spliced out of the MST4 mRNA transcript, could account for the sequence found in the truncated MST4a variant. These results suggest that MST4a is likely to be an alternatively spliced form of MST4. When we did a more comprehensive analysis of the tissue distribution of MST4 and MST4a by PCR, we were able to detect both forms in cDNAs from a variety of other adult and fetal human tissues (CLONTECH) including kidney, lung, liver, and pancreas as well as fetal thymus, spleen, muscle, liver, and kidney (Fig. 2, right panel). In most tissues, the major expressed form is MST4, but it appears that MST4a is more highly expressed in the brain. A human multiple tissue Northern blot was used to identify the expression pattern of MST4. Only one mRNA band with a size of 3.6 kilobases was detected. The results showed that MST4is ubiquitously expressed, with strong expression in placenta, weak expression in skeletal muscle and pancreas, and moderate expression in brain, heart, lung, liver, muscle, and kidney. It was not possible to determine the difference in expression of MST4 versus MST4a by this approach, since the difference in size between the two mRNAs is too small to be detected by Northern analysis (Fig.4). To map the MST4 gene, we performed fluorescence in situ hybridization using a biotin-labeled MST4 probe on normal human metaphase chromosomes. Hybridization of the MST4 cDNA probe resulted in specific labeling only of the X chromosome (Fig.5). Labeling of Xq25–27 was observed on four (eight cells), three (16 cells), or two (one cell) chromatids of the X chromosome homologues in 25 cells examined from mitogen-stimulated lymphocytes isolated from a healthy female. Of 82 signals observed, one signal (1.2%) was located at Xq25, 67 signals (82%) were located at Xq26, and 14 signals (17%) were located at Xq27. No background signals were observed at other chromosomal sites. We also observed a specific signal at Xq26 in an additional hybridization experiment using this probe (data not shown). These results indicate that the MST4 gene is localized to Xq26. This result was later verified by examination of the relative location of the MST4 gene in the X chromosome genomic map.Figure 5In situ hybridization of a biotin-labeled MST4 probe to human metaphase cells from phytohemagglutinin-stimulated peripheral blood lymphocytes.The X chromosome homologues are identified with arrows; specific labeling was observed at Xq26. The inset shows partial karyotypes of two X chromosome homologues illustrating specific labeling at Xq26 (arrows). Images were obtained using a Zeiss Axiophot microscope coupled to a cooled charge-coupled device (CCD) camera. Separate images of 4,6-diamidino-2-phenylindole dihydrochloride-stained chromosomes and the hybridization signal were captured and merged using image analysis software (IP LabSpectrum).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to determine whether MST4 or its variant is an active kinase, the N-terminal domains of MST4 and MST4a were tagged with a FLAG epitope, and the cDNAs were expressed in 293 cells using a cytomegalovirus promoter (Fig.6 A). To control for nonspecific kinase activity, a kinase-inactive mutant of MST4, MST4-K53R, was also transiently transfected into 293 cells. Following transfection, 293 cells were lysed, and the cell lysates were subjected to immunoprecipitation with anti-FLAG antibody (Fig. 6 B). The immunoprecipitates were then assayed for in vitro kinase assay using myelin basic protein as a substrate. The results indicate that MST4 is an active kinase, whereas MST4a did not exhibit kinase activity (Fig. 6 B). Interestingly, the kinase activity of MST4 isolated from cells grown in serum-free or serum-containing medium was comparable, indicating that MST4 possesses a high basal kinase activity (Fig. 6 C). Under the same conditions, a phosphoprotein with the same size as MST4 (46 kDa) was also detected in an in vitro kinase reaction when immunoprecipitates containing MST4 but not kinase-inactive MST4 (MST4-K53R) were used, suggesting that MST4 also functions as an autophosphorylating kinase (Fig. 6 C). Since MST4a was isolated by association with the Raf kinase domain in a yeast two-hybrid system, we determined whether MST4 or MST4a associate with c-Raf-1 in cells. Therefore, COS cells were co-transfected with expression vectors for Myc-Raf-1 and FLAG-MST4 or FLAG-MST4a. Raf was then immunoprecipitated with an anti-Myc antibody and analyzed for MST4 association by immunoblotting with an anti-FLAG antibody. Conversely, MST4 or MST4a was immunoprecipitated with an anti-FLAG antibody and analyzed for Raf association with an anti-Myc antibody. As shown in Fig. 6 D, no association of c-Raf-1 with MST4 or MST4a was observed. We also determined whether co-expression of Myc-Raf-1 with FLAG-MST4 altered MST4 kinase activity. Consistent with the co-immunoprecipitation results, no effect of Raf-1 on MST4 kinase activity was detected (data not shown). These results suggest that MST4 is not stably associated with or modulated by c-Raf-1. Removal of the C-terminal regulatory domain of MST1 and MST2 by caspases results in a significant increase in MST1 or MST2 kinase activity (35Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (325) Google Scholar, 40Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar). To address the function of the C-terminal tail of MST4, a cDNA encoding the N-terminal kinase domain of MST4 but lacking the C terminus of the protein (FLAG-MST4-NT) was transfected into COS cells. For comparison, COS cells were also transfected with expression vectors for FLAG-MST4 or FLAG-MST4-K53R. Following immunoprecipitation with anti-FLAG antibodies, the MST4 proteins were assayed for kinase activity using MBP as a substrate. As shown in Fig. 7, FLAG-MST4-NT has reduced kinase activity compared with MST4, indicating that the C-terminal domain acts to enhance MST4 activity. Interestingly, MST4-NT is no longer autophosphorylated, suggesting that the site of autophosphorylation may be in the C-terminal tail of the protein (Fig.7). MST4a was isolated by interaction with the Raf kinase domain and is most closely related to MST3, an enzyme that has been reported by one group to potentiate the Raf/MEK/ERK signaling cascade. To determine whether overexpression of MST4 might also activate ERK1 or ERK2, vectors expressing MST4, MST4-K53R or control vector were co-transfected with HA-ERK2 into 293 cells. The tagged ERK2 was immunoprecipitated from the lysates of transfected cells using an anti-HA antibody, and its kinase activity was assayed by Western blotting with anti-phospho-pTEpY MAPK antibody. A sample from each lysate was assayed directly by Western blotting with anti-FLAG antibody to confirm the expression of MST4 (data not shown). As shown in Fig. 8, overexpression of MST4 does not significantly affect the activation of ERK2 in cells grown either in serum or under serum-free conditions. Furthermore, we have also conducted similar experiments involving co-expression of MST4 (or MST4a or MST4-K53R), ERK2, and Myc-Raf-1 to determine whether MST4 can potentiate or inhibit Raf activation of ERK. Again, we could not detect a
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