Detection of a Novel Quiescence-dependent Protein Kinase
2000; Elsevier BV; Volume: 275; Issue: 33 Linguagem: Inglês
10.1074/jbc.m000818200
ISSN1083-351X
AutoresHwa‐Chain Robert Wang, Kellie A. Fecteau,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoWe have identified a cell quiescence-specific 33-kDa cytoplasmic protein kinase (p33QIK,Quiescence-Induced Kinase) based on induction of p33QIK-specific kinase activity of cells growth-arrested in the quiescent phase and deactivation upon entry into the cell cycle. Blockage of macromolecular synthesis prevents p33QIK from deactivation, indicating a requirement of newly synthesized regulators for deactivation of p33QIK during G0/G1 transition. Stress shock induces additional increases of p33QIK activity in a quiescence-dependent manner that correlates with induction of apoptosis. Using a specific antibody to Krs1/Mst2 protein, we found that p33QIK is related to p63Krs1 and is distinguishable from a 36-kDa protein kinase, which is induced through proteolytic modification of activated p63Krs1 in proliferating cells undergoing apoptosis. p33QIK is constantly expressed in quiescent, proliferating, and apoptotic quiescent cells. Regulation of p33QIK activity involves protein phosphorylation/dephosphorylation in a proteolysis-independent manner. Regulation of p33QIK and related p63Krs1 and p36 appears to involve distinct pathways in quiescent and proliferating cells, respectively. Our results illustrate the relevance of p33QIK activity for cell quiescence that may provide a new insight into signaling pathways regulated in cells during quiescence and quiescence-related apoptosis. We have identified a cell quiescence-specific 33-kDa cytoplasmic protein kinase (p33QIK,Quiescence-Induced Kinase) based on induction of p33QIK-specific kinase activity of cells growth-arrested in the quiescent phase and deactivation upon entry into the cell cycle. Blockage of macromolecular synthesis prevents p33QIK from deactivation, indicating a requirement of newly synthesized regulators for deactivation of p33QIK during G0/G1 transition. Stress shock induces additional increases of p33QIK activity in a quiescence-dependent manner that correlates with induction of apoptosis. Using a specific antibody to Krs1/Mst2 protein, we found that p33QIK is related to p63Krs1 and is distinguishable from a 36-kDa protein kinase, which is induced through proteolytic modification of activated p63Krs1 in proliferating cells undergoing apoptosis. p33QIK is constantly expressed in quiescent, proliferating, and apoptotic quiescent cells. Regulation of p33QIK activity involves protein phosphorylation/dephosphorylation in a proteolysis-independent manner. Regulation of p33QIK and related p63Krs1 and p36 appears to involve distinct pathways in quiescent and proliferating cells, respectively. Our results illustrate the relevance of p33QIK activity for cell quiescence that may provide a new insight into signaling pathways regulated in cells during quiescence and quiescence-related apoptosis. 33-kDa cytoplasmic protein kinase (Quiescence-Induced Kinase) Z-Asp-[(2,6-dichlorobenzoyl)oxy]methane c-Jun NH2-terminal kinase myelin basic protein actinomycin D polyacrylamide gel cycloheximide 12-O-tetradecanoylphorbol 13-acetate staurosporine FR901228 protein phosphatase 1 lambda protein phosphatase Many types of cells can remain healthy in a nonproliferative quiescent state in vivo for a long time (1Denhardt D.T. Edwards D.R. Parfett C.L. Biochim. Biophys. Acta. 1986; 865: 83-125PubMed Google Scholar, 2Cross F. Roberts J. Weintraub H. Annu. Rev. Cell Biol. 1989; 5: 341-396Crossref PubMed Scopus (133) Google Scholar). Normal cells in cultures that lack growth factors become growth-arrested in quiescent state (3Campisi J. Morreo G. Pardee A.B. Exp. Cell Res. 1984; 152: 459-466Crossref PubMed Scopus (59) Google Scholar, 4Larsson O. Zetterberg A. Engstrom W. J. Cell Sci. 1985; 75: 259-268PubMed Google Scholar). Stimulation of quiescent cells with growth factors transiently induces the mitogen-activated signaling pathway and the G0/G1 transition to drive cells to enter into the cell cycle (5Howe L.R. Leevers S.J. Gòmez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 6Dent P. Haser W. Haystead T.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (501) Google Scholar, 7Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1176) Google Scholar, 8Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (135) Google Scholar). The mitogen-activated signaling pathway, which comprises Ras, Raf, Mek, Erk, and Rsk, plays a critical role in induction of transcription factors and expression of the immediate early G1 gene products, leading to induction of secondary cellular events for completion of the cell cycle (7Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1176) Google Scholar, 9Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (828) Google Scholar, 10Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Science. 1995; 269: 403-407Crossref PubMed Scopus (884) Google Scholar). Constant activation of the mitogen-activated pathway leads to increased cell proliferation and induction of cellular transformation (5Howe L.R. Leevers S.J. Gòmez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 6Dent P. Haser W. Haystead T.J. Vincent L.A. Roberts T.M. Sturgill T.W. 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Although regulation of signaling pathways and gene expression in cells entering into different phases of the cell cycle have been heavily studied, the signaling pathway for growth arrest of cells or cells remaining in quiescent state has not been fully elucidated. Several growth arrest-specific (gas) genes have been reported based on their preferential expression in quiescent cultures and down-regulation upon entry of cells into the cell cycle (12Schneider C. King R.M. Philipson L. Cell. 1988; 54: 787-793Abstract Full Text PDF PubMed Scopus (841) Google Scholar, 13Ciccarelli C. Philipson L. Sorrentino V. Mol. Cell. Biol. 1990; 10: 1525-1529Crossref PubMed Scopus (73) Google Scholar, 14Manfioletti G. Ruaro M.E. Sal G.D. Philipson L. Schneider C. Mol. Cell. Biol. 1990; 10: 2924-2930Crossref PubMed Scopus (221) Google Scholar, 15Welcher A.A. Suter U. Leon M.D. Snipes G.J. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7195-7199Crossref PubMed Scopus (208) Google Scholar, 16Coccia E.M. Cicala C. Charlesworth A. Ciccarelli C. Rossi G.B. Philipson L. Sorrentino V. Mol. Cell. Biol. 1992; 12: 3514-3521Crossref PubMed Scopus (196) Google Scholar, 17Manfioletti G. Brancolini C. Avanzi G. Schneider C. Mol. Cell. Biol. 1993; 13: 4976-4985Crossref PubMed Scopus (539) Google Scholar). However, the significance of these gene products in signaling control of cell quiescence is unclear. While investigating the signaling control regulated by protein kinases during the transition between G0 and G1 phases, we detected a novel cytoplasmic protein kinase of 33 kDa (p33QIK)1 by using the in-gel kinase assay with myelin basic protein (MBP) as a substrate. On the basis of p33QIK activation in cultures arrested in quiescent state and its deactivation in cultures entering into G1 phase of the cell cycle, it appears that p33QIK is involved in signaling control for cell quiescence. Deactivation of p33QIK requires expression of the immediate early G1 gene products, such as protein phosphatases. Stress shock may additively increase p33QIKactivity in correlation with induction of apoptosis in cultures arrested in quiescent state but not in the other phases of the cell cycle. Using specific antibodies we determined that p33QIKis a gene product of the Krs1/Mst2 gene (18Wang H.-C.R. Erikson R.L. Mol. Biol. Cell. 1992; 3: 1329-1337Crossref PubMed Scopus (56) Google Scholar, 19Creasy C.L. Chernoff J. Gene (Amst.). 1995; 167: 303-306Crossref PubMed Scopus (119) Google Scholar, 20Taylor L.K. Wang H.-C.R. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar). We also found that p33QIK is related to, but distinguishable from, a protein kinase of 36 kDa (p36), which is proteolytically derived from either Krs1/Mst2 or Krs2/Mst1 in cells undergoing apoptosis (21Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (328) Google Scholar, 22Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar, 23Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 24Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar). In this communication, our results not only reveal a tight correlation between induction of the specific kinase activity of p33QIK and growth arrest of cells in quiescence, but also a correlation between distinct activation levels of p33QIK and induction of apoptosis of cells growth-arrested in quiescent state. Because this is the first report describing a protein kinase that is tightly regulated throughout the cell cycle and the kinase activity is specifically induced in cell quiescence, our report provides an important insight into the signaling cascade for regulation of molecular processes in cells growth-arrested in the quiescent state. Mouse NIH3T3 fibroblast cultures were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin, and cultivated at 37 °C. Mouse 10T1/2 fibroblast cultures were maintained in Basal medium Eagle Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 5 μg/ml streptomycin, and cultivated at 37 °C. Cells synchronized in quiescent state were growth-arrested by serum starvation for 48 h. Actinomycin D (ActD), cycloheximide (CHXM), 12-O-tetradecanoylphorbol 13-acetate (TPA), staurosporine (STSP) (Sigma), sodium fluoride, sodium orthovanadate (Na3VO4) (Fisher Scientific, Pittsburgh, PA), and FR901228 (FR) (National Cancer Institute, Frederick, MD) were diluted in culture medium before treatment of cultured cells. Production of rabbit polyclonal antibody Ab-KQ to p63Krs1and p33QIK was made using peptide sequences of Krs1 as antigens (Alpha Diagnostic International, San Antonio, TX). UV irradiation was carried out using a UV cross-linker (Stratagene, La Jolla, CA). After rinsing with phosphate-buffered saline, cells were trypsinized from culture dishes, rinsed with Ca2+ and Mg2+ free phosphate-buffered saline, fixed in 70% cold ethanol, and stained with 10 μg/ml propidium iodide for 30 min (25Darzynkiewicz Z. Robinson J.P. Crissman H.A. Method in Cell Biology : Flow Cytometry, 2nd Ed., Part A. Academic Press, San Diego, CA1994Google Scholar). Flow cytometry analysis was performed on the Coulter EPICS Elite cytometer (Hialeah, FL) using a 15-milliwatt air-cooled argon laser producing 488-nm light in which propidium iodide fluorescence light emission is collected with a 610LPDC filter. Extended analysis of DNA content and calculation of the percentage of cells in each phase of the cell cycle were performed on Multicycle software (Phoenix Flow System, San Diego, CA). Cells were lysed with 30 strokes of a tight-fitting Dounce homogenizer in lysis A buffer (10 mm KH2PO4, 1 mm EDTA, 5 mm EGTA, 10 mm MgCl2, 50 mm β-glycerolphosphate, 1 mmNa3VO4, 2 mm dithiothreitol, pH 7.2) (18Wang H.-C.R. Erikson R.L. Mol. Biol. Cell. 1992; 3: 1329-1337Crossref PubMed Scopus (56) Google Scholar). Cytoplasmic proteins (S20) were isolated from the supernatants after centrifugation of crude lysates at 20,000 ×g for 20 min. Protein concentration in S20 was measured using the BCA assay (Pierce). Immunoprecipitation of p63Krs1 and p33QIK was carried out by incubation of S20 with the specific antibody Ab-KQ at 0 °C for 1 h. Immune complexes were adsorbed to Pansorbin (Staphylococcus aureus) (Calbiochem, La Jolla, CA) at 0 °C for 30 min and washed with STE buffer (50 mmTris-HCl, pH 7.0, 150 mm NaCl, 1 mmNa2EDTA) supplemented with 0.1 mmdithiothreitol and 0.1% Nonidet P-40 and ST buffer (10 mmTris-HCl, pH 7.2, 150 mm NaCl) supplemented with 1 mm dithiothreitol. MBP (Life Technologies, Inc.) was used as a substrate in the assay, and the in-gel kinase assay was performed as described previously (18Wang H.-C.R. Erikson R.L. Mol. Biol. Cell. 1992; 3: 1329-1337Crossref PubMed Scopus (56) Google Scholar). Briefly, 10% SDS-polyacrylamide gel (SDS-PAG) was copolymerized with 0.4 mg/ml MBP. Cellular proteins were resolved in the MBP-immobilized SDS-PAG, followed by rinsing the gel with 20% isopropanol in buffer B (100 mm Tris, pH 8, 5 mm β-mercaptoethanol). The gel was denatured in buffer B supplemented with 6 m guanidine-HCl and renatured with buffer B supplemented with 0.04% Tween 40 (26Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (450) Google Scholar). The kinase reaction was carried out by incubating the gel with kinase buffer (20 mm Tris, pH 7.2, 10 mm MgCl2, 15 mm β-glycerolphosphate) supplemented with 50 μm γATP and 50 μCi of [γ-32P]ATP at 22 °C for 30 min. The gel was washed with 1% sodium pyrophosphate in 5% trichloroacetic acid. Protein kinases, which phosphorylated MBP in the gel, were detected by autoradiography of the γ-32P-labeled MBP. Proteins were resolved by electrophoresis in a 10% SDS-PAG and transferred to a nitrocellulose filter (pore size: 0.4 μm) (Life Technologies, Inc.). Nonspecific protein binding sites on the filter were saturated by incubation of the filter with 3% nonfat milk in ST buffer at ambient temperature for 30 min. Filters were then incubated with the specific primary antibody at 4 °C for 15 h. The filters were rinsed three times and incubated with horseradish peroxidase-conjugated antibodies at ambient temperature for 30 min. Antigen-antibody complexes on filters were detected by the Supersignal chemiluminescence kit as indicated by the manufacturer (Pierce) and visualized by autoradiography. Protein phosphatase 1 (PP1) and lambda protein phosphatase (λPPase) were purchased from New England BioLabs (Beverly, MA). Protein substrate was incubated with 0.1 unit of PP1 or 100 units of λPPase in phosphatase buffer (50 mm Tris-HCl, pH 7.0 or 7.5, 0.1 mmEDTA, 5 mm dithiothreitol, 0.01% Brij-35, 2 mmMnCl2) at 30 °C for 30 min. Recombinant human Caspase-3 was purchased from Alexis Biochemicals (San Diego, CA). Substrate was incubated with 0.2 μg of caspase-3 in reaction buffer (10 mm KH2PO4, 1 mm EDTA, 5 mm EGTA, 10 mm MgCl2, 50 mm β-glycerolphosphate, 1 mmNa3VO4, 2 mm dithiothreitol, pH 7.2) at 37 °C for 30 min. A broad range caspase inhibitorZ-Asp-[(2,6-dichlorobenzoyl)oxy]methane (Z-d-CH2-DCB) (27Dolle R.E. Hoyer D. Prasad C.V. Schmidt S.J. Helaszek C.T. Miller R.E. Ator M.A. J. Med. Chem. 1994; 37: 563-564Crossref PubMed Scopus (177) Google Scholar, 28Mashima T. Naito M. Fujita N. Noguchi K. Tsuruo T. Biochem. Biophys. Res. Commun. 1995; 21: 1185-1192Crossref Scopus (204) Google Scholar) (Calbiochem) was dissolved in dimethyl sulfoxide and diluted in reaction buffer. By the in-gel kinase assay using MBP as a substrate, we detected a cytoplasmic protein kinase with a molecular mass of 33 kDa, p33QIK, in different types of cells, including human epithelial cells (29Rajgolikar G. Chan K.K. Wang H.-C.R. Breast Cancer Res. Treat. 1998; 51: 29-38Crossref PubMed Scopus (67) Google Scholar) and mouse fibroblasts. The kinase activity of p33QIK was closely up-regulated in mouse fibroblast NIH3T3 and 10T1/2 cells in the quiescent state, which had been induced by serum starvation. Activity of p33QIK was down-regulated in cells entering the cell cycle of mitosis. Investigating the regulation of p33QIK activity in the cell cycle, NIH3T3 cultures were growth-arrested and synchronized in quiescent state by serum starvation for 48 h. After 48 h of serum starvation, 90% of cells in the NIH3T3 culture remained in G0/G1 phase (Fig. 1 A, panel a); concomitantly, induction of p33QIK activity was detected (Fig. 1 B, lane 1). Synchronized quiescent cells were then stimulated with serum to enter the cell cycle. In one hour of serum stimulation of quiescent cultures, transient induction of p44/42Erk activity, an index of the mitogen-activated signaling pathway, was detected (Fig. 1B, lanes 2 and3) as normally happens during the G0/G1 transition. After 6 h of serum stimulation, p33QIK activity decreased to a basal level (Fig. 1 B, lane 4) while a major population of cells was still in G1 phase (Fig. 1 A,panel b). After 18 h of serum treatment, a major population of cells entered the S phase (Fig. 1 A,panel c) and p33QIK remained inactive (Fig.1 B, lane 5). Cultures were then deprived of serum for a second time to synchronize cells in quiescence (Fig.1 B, lanes 6–9). Between 4 and 8 h after initiation of the second serum starvation, 22 and 26 h after the first entry of cells into the cell cycle, mitosis was observed microscopically. Also, between these two time points, a significant population of cells was detected in G2/M phase by flow cytometry (Fig. 1 A, panel d), and p33QIK activity was gradually induced in cultures entering G0 phase from M phase (Fig. 1 B, lane 7). After 12 h of the second serum starvation the major population of cells was arrested in G0 state (Fig.1 A, panel e), and p33QIK activity reached a maximal level (Fig. 1 B, lane 8). p33QIK remained active in quiescent cultures (Fig.1 B, lane 9) following the extended 48-h serum starvation (Fig. 1 A, panel f). Serum stimulation of these secondly arrested quiescent cultures consistently induced a transient activation of p44/42Erk prior to deactivation of p33QIK (Fig. 1 B, lanes 10 and11). Induction of the kinase activity of p33QIKin cells was closely correlated to growth arrest in quiescent state, whereas deactivation of p33QIK was correlated with entry of cells into the cell cycle and induction of p44/42Erkactivity. It appears that down-regulation of p33QIKcorrelates with activation of the mitogen-activated signaling pathway. By subcellular fractionation, p33QIK activity was mainly detected in the cytoplasmic compartment of quiescent cells and cells entering into G1 phase of the cell cycle (data not shown). We found that p33QIK may be a gene product of theKrs1/Mst2 gene. Using a Krs1-specific antibody (Ab-KQ) in Western immunoblotting, we detected that p33QIKwas constantly expressed in cultures entering into different phases of the cell cycle (Fig. 1 C). Changes of p33QIKactivity in cells undergoing the cell cycle appear to result from regulation of the specific kinase activity. To study deactivation kinetics of p33QIK in cultures by induction of p44/42Erk activity, we stimulated quiescent NIH3T3 cultures with different reagents (serum, TPA, sodium vanadate, okadaic acid, and sodium fluoride) to induce p44/42Erk kinase activity. In comparison with serum stimulation of p44/42Erk activity (Fig. 2, lanes 2–4), treatment of quiescent NIH3T3 cultures with sodium vanadate, which acts as a general inhibitor of tyrosine phosphatases, resulted in a profound and constant activation of p44/42Erk (Fig. 2, lanes 5–7). Concomitantly, an accelerated deactivation of p33QIK was induced in cells treated with sodium vanadate (Fig. 2, lanes 5–7) as compared with cells stimulated with serum (Fig. 2, lanes 2–4). Cultures stimulated with phorbol ester (data not shown) exhibited similar kinetics to cells stimulated with serum in regulation of p33QIK and p44/42Erk activity; there was a progressive decrease of p33QIK activity and a transient induction of p44/42Erk activity. Consistent with a previous observation (18Wang H.-C.R. Erikson R.L. Mol. Biol. Cell. 1992; 3: 1329-1337Crossref PubMed Scopus (56) Google Scholar), treatment of cells with okadaic acid activated p63Krs1 and treatment of cells with sodium fluoride-induced p44/42Erk activity. Also, sodium fluoride treatment resulted in deactivation of p33QIK, but okadaic acid treatment did not result in deactivation of p33QIK (data not shown). Down-regulation of p33QIK activity was associated tightly with induction of the immediate early G1signaling pathway indexed by activation of p42/44Erk. To investigate whether the immediate early G1 gene product is required to down-regulate p33QIK activity, we treated cells with ActD, an inhibitor of gene transcription, or CHXM, an inhibitor of protein synthesis. Treatment of cultures with ActD (Fig. 3,lanes 4 and 5) or CHXM (Fig. 3, lanes 6 and 7) blocked the serum-induced down-regulation of p33QIK activity (Fig. 5, lanes 2 and3). Treatment with phorbol ester TPA and ActD (Fig. 3,lanes 10 and 11) or CHXM (Fig. 3, lanes 12 and 13) resulted in not only blockage of the TPA-induced deactivation of p33QIK but also in an increase in the kinase activity of p33QIK (Fig. 3, lanes 10–13). In addition, in the presence of ActD or CHXM, treatment of cells with serum or TPA resulted in a constant activation of p44/42Erk (Fig. 3, lanes 4–7 and10–13). Treatment with ActD or CHXM appeared to block deactivation of p33QIK and p44/42Erk in cultures after the stimulation with serum or TPA. Activation and deactivation of p44/42Erk are known to result from protein phosphorylation by Mek (30Payne D.M. Rossomando A.J. Martino P. Erikson A.K. Her J. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (871) Google Scholar, 31Pelech S.T. Sanghera J.S. Science. 1992; 257: 1355-1356Crossref PubMed Scopus (308) Google Scholar, 32Crews C.M. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (743) Google Scholar) and dephosphorylation by phosphatases such as MKP-1, which is an immediate early G1 gene product (33Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1040) Google Scholar, 34Zheng C.-F. Guan K.L. J. Biol. Chem. 1993; 268: 16116-16119Abstract Full Text PDF PubMed Google Scholar), respectively. Blocking expression of the immediate early G1 gene products by ActD or CHXM protected p44/42Erk from deactivation. Conceivably, deactivation of p33QIK may also require expression of an immediate early G1 gene product. In the absence of serum, treatment of quiescent cultures with either ActD or CHXM resulted in significant increases of p33QIK activity (Fig. 3, lanes 14–17) while apoptotic-like cell death occurred in these cultures. Profound activation of p33QIK appears to correlate with induction of apoptosis in quiescent cultures.Figure 5Determination of p33QIK identity. NIH3T3 cultures were synchronized in quiescent state by serum starvation for 48 h (G0), followed by treatment with 300 mm NaCl (G0/S) for 2 h. Growing NIH3T3 cultures were maintained in complete Dulbecco's modified Eagle's medium with 5% serum and treated with 25 nm STSP (G/STSP) for 48 h (B) or 24 h (C). The immune complexes of p63Krs1, p36, and p33QIK were prepared from 10 μg of S20 incubated with 1 μl of Ab-KQ (IP). A, the kinase activities of p63Krs1and p33QIK in 10 μg of S20 isolated from G0 (lane 1) or G0/S (lane 4) and in the immune complexes (IP) in the absence (lanes 2 and 5) or presence of 1 μg of antigen peptides (lanes 3 and 6) were determined.B, the kinase activities of p63Krs1, p36, and p33QIK in 10 μg of S20 (lanes 1 and2) and in the immune complexes (IP) (lanes 3 and 4) isolated from G0/S (lanes 1 and 3) or G/STSP (lanes 2 and4) were determined. C, the immune complexes of p63Krs1, p36, or p33QIK isolated from cultures of G0/S (lanes 2–4) and G/STSP (lanes 6–8) were treated with 0.2 μg caspase-3 (C3)(lanes 3, 4, 7, and8) in the presence of 5 μm caspase inhibitorZ-d-CH2-DCB (I) (lanes 4 and 8) at 37 °C for 30 min. The kinase activities of p63Krs1, p36, and p33QIKwere determined by the in-gel kinase assay using MBP as a substrate.Bars indicate p63Krs1, p36, and p33QIK.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Several groups of investigators reported that activation of a 33- or 36-kDa MBP kinase in cancer cells correlated with induction of apoptosis by anticancer agents or stress shock (22Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar, 23Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 24Watabe M. Kakeya H. Osada H. 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To investigate whether activation of p33QIK was a stress-related event, the kinase activity of p33QIK was measured in cultures entering into different phases of the cell cycle and undergoing stress shock. NIH3T3 cultures were growth-arrested in quiescent phase by serum starvation (Fig.4, A and B,lanes 1). Quiescent cultures were released by serum stimulation for 1 or 20 h, to enter G1 or G2/M phase of the cell cycle, respectively. Cultures were then treated with 100 mJ/cm2 of UV irradiation and incubated for another 1 or 3 h (Fig. 4 A). The kinase activity of p33QIK was elevated in quiescent cultures by UV irradiation in a time-dependent manner (Fig. 4 A,lanes 2 and 3). Increases in activation of p33QIK by UV irradiation were also dose-dependent (data not shown). In contrast, p33QIK activity was not induced by UV irradiation in cultures in which a major population of cells was in either G1 or G2/M phase (Fig. 4 A,lanes 5 and 7). Similarly, osmotic shock with 300 mm NaCl induced a profound activation of p33QIKin quiescent cultures (Fig. 4 B, lane 2) but did not induce p33QIK activity in cultures entering into G1 or G2/M phase (Fig. 4 B,lanes 4 and 6). Cultures containing profoundly elevated p33QIK activity exhibited apoptotic morphology with nuclear condensation and cell shrinkage. Addition of serum into cultures immediately after the shock of stress did not suppress apoptotic phenotype or prevent p33QIK from activation (data not shown). The highly elevated p33QIK activity induced by stress shock appears to be involved in induction of apoptosis in a quiescence-dependent manner. Initially, during chromatographic purification of p33QIK, we observed coelution of p63Krs1 and protein kinases with different molecular masses from 55 to 33 kDa. Lately, several groups reported that the proteolysis of Krs/MST proteins by caspases generated a 36-kDa protein kinase (21Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (328) Google Scholar, 22Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar, 23Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 24Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar). By using peptides derived from Krs1 sequences as antigens to generate antibodies, we obtained an antibody designated as Ab-KQ that interacted with both p63Krs1 and p33QIK in assays of immunoprecipitation and immunoblotting. The Ab-KQ was generated by immunization of rabbits with a Krs1 peptide containing the residue sequence EIKAKRHDEQQRELEE, which has 50% homology with a region of Krs2/Mst1 (20Taylor L.K. Wang H.-C.R. Erikson R.L. Proc. Natl. Acad. Sci. U. S. 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