The Cyclin-dependent Kinase Inhibitor p27Kip1 Is Stabilized in G0 by Mirk/dyrk1B Kinase

Abstract

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G0/G1 until mitogenic signals activate G1 cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G0 in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G0-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G0, but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNAi to Mirk increased cell entry from G0 into G1 as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G0 when Mirk is most abundant. Mitogen stimulation then causes cells to enter G1, reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNAi in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation. Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G0/G1 until mitogenic signals activate G1 cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G0 in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G0-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G0, but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNAi to Mirk increased cell entry from G0 into G1 as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G0 when Mirk is most abundant. Mitogen stimulation then causes cells to enter G1, reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNAi in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation. The cyclin-dependent kinase inhibitor p27 (1Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar, 2Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar) plays a critical role in regulating progression through the cell cycle. Rising levels of p27 block the cell in G0/G1 until mitogenic signals activate G1 cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. CDK2/cyclin E phosphorylates p27 at Thr-187 (3Sheaff R. Groudine M. Gordon M. Roberts J. Clurman B. Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (797) Google Scholar, 4Muller D. Bouchard C. Rudolph B. Steiner P. Stuckman I. Saffrich R. Ansorge W. Huttner W. Eilers M. Oncogene. 1997; 15: 2561-2576Crossref PubMed Scopus (149) Google Scholar) and promotes its degradation by the ubiquitin-proteasome pathway at the G1-S checkpoint (5Pagano M. Tam S. Theodoras A. Beer-Romano P. Del Sal G. Chau V. Yew P. Draetta G. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1736) Google Scholar). Mitogenic signals also induce the kinase KIS, which mediates phosphorylation of p27 at Ser-10 (6Boehm M. Yoshimoto T. Crook M. Nallamshetty S. True A. Nabel G. Nabel E. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (247) Google Scholar), facilitating the translocation of p27 to the cytoplasm at later times in G1 (7Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 8Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.-I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). However, p27 has been shown to be already phosphorylated at Ser-10 in G0/early G1 before KIS induction (7Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 8Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.-I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Furthermore, p27 is solely nuclear in G0 and does not even bind to its exportin CRM1 until cells enter G1 following mitogen stimulation (9Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar), implying that phosphorylation at Ser-10 may fulfill another function. In this study we identify the arginine-directed serine/threonine kinase Mirk/dyrk1B as a kinase active in G0 which phosphorylates p27 at Ser-10 without inducing its translocation from the nucleus. Mirk/dyrk1B is a member of the Dyrk/minibrain family of arginine-directed serine/threonine kinases. Mirk is transcriptionally up-regulated by Rho family members (10Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and itself functions as a transcriptional activator (11Lim S. Jin K. Friedman E. J. Biol. Chem. 2002; 277: 25040-25046Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) under control of the MKK3/p38 MAPK signaling system (11Lim S. Jin K. Friedman E. J. Biol. Chem. 2002; 277: 25040-25046Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Lim S. Zou Y. Friedman E. J. Biol. Chem. 2002; 277: 49438-49445Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Mirk is expressed at elevated levels in some tumor cells and in normal skeletal muscle (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar) where it is active in muscle differentiation (10Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). However, Mirk is widely expressed at low levels, suggesting it performs some general role. In the current study we demonstrate a novel function for Mirk, stabilization of the CDK inhibitor p27 kip1 during the G0 phase of the cell cycle. Materials—Antibody to p27Kip1 was from Transduction Laboratories; antibodies to PCNA, 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; EGFP, enhanced green fluorescent protein; DTT, dithiothreitol; PBS, phosphate-buffered saline; GST, glutathione S-transferase; CDK, cyclin-dependent kinase; HNF1α, hepatocyte nuclear factor 1α. CDK2, β-tubulin, and cyclin A were from Santa Cruz Biotechnology, and antibody to the FLAG-epitope was from Sigma. Antibody to the Ser-10-phosphorylated form of p27 was purchased from Zymed Laboratories Inc.. Affinity-purified rabbit polyclonal antibody to a unique sequence at the C terminus of Mirk was raised as described (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). In some experiments affinity-purified rabbit polyclonal antibody to the unique N terminus of Mirk was used (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). GST-ERK2 was purchased from Upstate Biotechnology, inc. Polyvinylidene difluoride transfer paper Immobilon-P was purchased from Millipore. PLUS reagent and LipofectAMINE were from Invitrogen. All radioactive materials were purchased from PerkinElmer Life Sciences, ECL reagents from Amersham Biosciences, and tissue culture reagents from Mediatech (Fisher). All other reagents were from Sigma. Cell Culture—C2C12 mouse myoblasts and NIH3T3 cells were obtained from the ATCC. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. C2C12 cells were maintained in growth medium: Dulbecco's modified Eagle's medium containing 20% fetal calf serum, and switched to differentiation medium/Dulbecco's modified Eagle's medium containing 2% horse serum. NIH3T3 and C2C12 cells were transfected with LipofectAMINE/PLUS according to the supplier's manual. Plasmids—pcDNA-Mirk, pcDNA-MirkYF, and pcDNA-MirkKR (kinase-inactive Mirk) had been generated previously (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). pGEX2T p27-wild-type, pGEX2T p27-NADA, pGEX2T p27-VPKK, and pGEX2T p27-NADA/VPKK were the kind of B. J. S. B. EMBO J. 1997; PubMed Scopus Google Scholar). p27Kip1 was by system from and into to All mutant p27 were to the RNAi to Mirk were and another sequence mutant at to Mirk, as the mutant was into the of p27 was from cell and the was with of in the of X. S. Friedman E. Cell Google Scholar). In were times with and times with kinase for at with of kinase containing of and of p27 protein as and then by and were as previously (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). and the p27 were into the and expressed and as described J. B. PubMed Scopus Google Scholar). of was with 10% of culture of cells the expression and at for expression was with for at and were by and at were in of containing and and for was to a of and then was to a of were by a for by of were by at for and of inhibitor were to the and were with of at with were times with of used for in kinase were from the by at with of glutathione DTT, of p27 used in kinase was from by with Biosciences, of in for at was from the by with of for at as and with PBS, cells were in 2% 10% were for and were protein reagent from the of cell were difluoride by The were in in and for at and for at with antibody in and were by enhanced of RNA from cell was in a to by and by The were to a p27 containing of p27 sequence in (1Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J. Tempst P. Massague J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar). were with by The was at with at of the at for with then for at in and were a and was the Mirk a G0 p27 in serine/threonine kinase Mirk/dyrk1B is widely expressed at low levels in normal (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), suggesting that Mirk may a general role in cell cell In addition, Mirk protein levels to widely in to of cells with and with (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), we Mirk regulation the cell cycle. NIH3T3 cells in G0/G1 when to and into phase in a N. Y. Mol. Cell. Biol. 1997; PubMed Google Scholar). levels of cyclin A were used to cell progression through G1 to S. Mirk protein levels were elevated in NIH3T3 cells, and levels when cells G1 and phase Mirk levels were elevated in G0/G1 and were in cells. of the CDK inhibitor p27 elevated when cells and in G0 S. W. J. Roberts J. Science. PubMed Scopus Google Scholar, K. M. C. Massague J. Roberts J. Koff A. Genes Dev. 1994; PubMed Scopus Google Scholar), the cell phase in which Mirk was we the that Mirk phosphorylated p27 in Mirk was from NIH3T3 cells and from cells at times and then its to p27 was by in kinase The phosphorylation of p27 by Mirk was in cells in G0/G1 with the Mirk levels later cells had the as shown by the in cyclin and the levels and the of Mirk to p27 Mirk p27 in and in at Ser-10 the at which Mirk phosphorylated p27. Mirk is a active kinase (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), its to p27 was in kinase Mirk phosphorylated the CDK inhibitor p27, with as the control In and had kinase p27 not phosphorylation at Thr-187 the and degradation of p27 by the (3Sheaff R. Groudine M. Gordon M. Roberts J. Clurman B. Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (797) Google Scholar, 4Muller D. Bouchard C. Rudolph B. Steiner P. Stuckman I. Saffrich R. Ansorge W. Huttner W. Eilers M. Oncogene. 1997; 15: 2561-2576Crossref PubMed Scopus (149) Google Scholar), we of the the Thr-187 phosphorylation the Ser-10 which the translocation of p27 to the cytoplasm N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.-I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, G. A. L. P. Draetta M. S. EMBO J. PubMed Scopus Google Scholar), Mirk The mutant a in with J. S. B. EMBO J. 1997; PubMed Scopus Google Scholar), a p27 mutant at its CDK and a mutant were phosphorylated by Mirk to a as wild-type p27 were to Mirk in Mirk is a Mirk was to it phosphorylated wild-type p27, and the p27 CDK phosphorylation mutant to a in in kinase In phosphorylated of the mutant p27 to less than wild-type p27. These that Mirk has a for the Ser-10 in p27 and suggest that Mirk is at of the for phosphorylation at Ser-10 in phosphorylates p27 at Ser-10. were of wild-type p27, p27 to GST-ERK2 was to p27 in in kinase The phosphorylated p27 were by and of p27 were Ser-10 is the phosphorylation for p27 in for of the amount of p27 phosphorylation as by with (7Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). of p27 at Ser-10 has been by of a p27-S10D phospho-mimetic to p27 translocation to the cytoplasm in to of Ser-10 to to the cytoplasm N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.-I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, G. A. L. P. Draetta M. S. EMBO J. PubMed Scopus Google Scholar). In G0, p27 is solely nuclear and does not even bind to its exportin CRM1 until cells enter G1 following mitogen stimulation (9Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar). These that phosphorylation of p27 at Ser-10 is a control in the mitogenic The kinase KIS has been as kinase that mediates phosphorylation of Ser-10 in (6Boehm M. Yoshimoto T. Crook M. Nallamshetty S. True A. Nabel G. Nabel E. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (247) Google Scholar). However, p27 has been shown to be phosphorylated already at Ser-10 to some in G0/early G1 before KIS induction (7Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 8Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.-I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Mirk is in NIH3T3 cells in G0 and it is by (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). These suggest that Mirk p27 at Ser-10 during G0, before mitogen induction of that Mirk phosphorylates p27 in wild-type was with wild-type p27-S10A expression into NIH3T3 cells. Following the were for with and then the p27 was with antibody and by and The amount of into wild-type p27 was when wild-type Mirk was was when wild-type p27 was with Mirk In of wild-type Mirk did not the low of into mutant p27-S10A for transfected p27 antibody expression of These data that Mirk phosphorylates p27 in at Ser-10. protein Mirk phosphorylation of p27 in its for wild-type p27 and wild-type Mirk, the were into NIH3T3 cells and to and the of p27 was by the p27 with wild-type Mirk a in wild-type Mirk was with the mutant p27 of the the The is itself a than wild-type p27 its was by with the that the p27 was by phosphorylation p27 were with calf before with the the p27 by wild-type Mirk, that Mirk phosphorylates p27 in solely at Ser-10. p27 at Ser-10 by Mirk to p27 phosphorylated at Ser-10 function as a CDK and Mirk were in NIH3T3 cells. Following phosphorylation by Mirk in p27 was capable of binding to The to decreased following with Mirk that it had been The amount of of antibody were in the These data demonstrate that p27 phosphorylated by Mirk in G0 was capable of as a CDK Mirk of p27 by RNAi to of endogenous Mirk to p27 at Ser-10 was by expression of RNA to Mirk the the Mirk were to Mirk and and the RNAi a mutant sequence was NIH3T3 cells were in medium with a to of RNAi to and with expression for p27 to the phosphorylation of p27 to be Cell levels of green fluorescent were for Mirk by with a in endogenous Mirk protein levels and in phosphorylation of p27 at Ser-10 was by Mirk p27 phosphorylation was with the RNA that was used as RNA control A of the and the of p27 phosphorylated at Ser-10 to p27 from and a that RNAi and endogenous Mirk levels and phosphorylation by was a of Mirk protein levels and of p27 phosphorylation at Ser-10. Mirk of p27 in of p27 at Ser-10 has been to p27 (7Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). We this by the of non-phosphorylatable phospho-mimetic and wild-type in NIH3T3 cells in G0 by by of by that the p27-S10D phospho-mimetic was the most stable Mirk levels with RNAi also decreased the stability of endogenous p27 Mirk by RNA Cell data that endogenous Mirk in G0 p27 and some cells to into this NIH3T3 cells were transfected for with were cultured for another to block the cells in G0 and to cells of Mirk enter G1 in the of Depletion of Mirk the stabilization of p27 and p27 RNAi to Mirk decreased Mirk levels to a of of control and decreased p27 levels by of the from G0 to G1 was by of the of the by RNAi to Mirk increased the of of the G0-arrested cell of Mirk p27 and the cell into Mirk with p27 in of p27 at Ser-10 has been shown to p27 translocation from the nucleus to the cytoplasm in to of Ser-10 to to the cytoplasm N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.-I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, G. A. L. P. Draetta M. S. EMBO J. PubMed Scopus Google Scholar). Mirk phosphorylates p27 at Ser-10 in G0, p27 remains in the nucleus until the of We the that phosphorylation at was not to initiate p27 translocation in G0 by with in G0-arrested NIH3T3 cells. of Mirk p27 and a were Mirk and were nuclear with some in the p27 and p27-S10A were solely in the nucleus. the were p27, wild-type and nuclear Mirk, wild-type phosphorylation of by Mirk was not to induce translocation of p27 from the nucleus to the cytoplasm in of by Mirk in G0 stabilizes p27 and increases its than facilitating its translocation of the nucleus. These data with the that Mirk in cell through phosphorylation of p27 at which occurs in G0 when Mirk is most abundant. Mitogen stimulation then causes cells to enter G1, reduces Mirk levels (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), and the KIS kinase that phosphorylates p27 during G1 (6Boehm M. Yoshimoto T. Crook M. Nallamshetty S. True A. Nabel G. Nabel E. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (247) Google Scholar). of Mirk during p27 to in used RNAi in C2C12 myoblasts to the of Mirk expression p27 stability in a cell system in which Mirk in differentiation. We (10Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) that depletion of endogenous Mirk from C2C12 myoblasts RNA interference the induction of and and the of myoblasts into We that of Mirk levels by RNA interference in myoblasts a in p27 protein levels with a 20% in p27 mRNA levels The of Mirk p27 mRNA levels was also by not and D. Lee K. Deng X. S. Friedman E. J. 2003; PubMed Scopus (25) Google Scholar), which that Mirk did not the of a p27 These data that Mirk plays a role in p27 stability in myoblasts as as NIH3T3 cells. The current study some in the was previously (7Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) that phosphorylation of p27 at Ser-10 increased stability p27, data which we in the current study NIH3T3 cells in N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.-I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar) data that phosphorylation of p27 at Ser-10 is for p27 to bind to the exportin CRM1 and to be to the cytoplasm during G1 where p27 is by the and less that phosphorylation of p27 at Ser-10 has which cell of p27 at Ser-10 during G0 by Mirk/dyrk1B stabilizes p27 and p27 the nucleus where it bind to CDK2. In phosphorylation of p27 at Ser-10 by the KIS kinase in G1 p27 to bind to CRM1 and to be into the cytoplasm for (6Boehm M. Yoshimoto T. Crook M. Nallamshetty S. True A. Nabel G. Nabel E. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (247) Google Scholar). In of other (9Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar) shown that of p27 with CRM1 is in G0 and increases during the G1 to phase different to the Mirk and KIS different transcriptional regulation not the phase of the cell cycle. transcriptionally Mirk/dyrk1B (10Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), induce the of KIS (6Boehm M. Yoshimoto T. Crook M. Nallamshetty S. True A. Nabel G. Nabel E. EMBO J. 2002; 21: 3390-3401Crossref PubMed Scopus (247) Google Scholar). Mirk levels and in G0 and during G1 when KIS levels and KIS is not during G0 to p27. The current study has shown that p27 is not to the cytoplasm even phosphorylation at Ser-10 by phosphorylation at Ser-10 is not to p27 to bind to CRM1 during Mirk and p27 a different of the nucleus than CRM1 during Mirk is in the nucleus with the protein S. Zou Y. Friedman E. J. Biol. Chem. 2002; 277: 49438-49445Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, Y. S. Lee K. Deng X. Friedman E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). functions as CRM1 and p27 K. Y. Y. S. M. T. Yoshida M. N. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The nuclear not been but we in study that not Y. S. Lee K. Deng X. Friedman E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), which is the and Y. S. Lee K. Deng X. Friedman E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. Y. S. M. T. Yoshida M. N. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The serine/threonine kinase Mirk/dyrk1B is a that functions in muscle differentiation (10Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) but is widely expressed at low levels, suggesting that it in some general control In the current study we Mirk phosphorylation of p27 to the of the cell in members of the Dyrk/minibrain family which has been in through its phosphorylation of protein H. W. J. A. A. C. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). functions in cells to cell through of the pathway L. P. PubMed Scopus Google Scholar, K. S. V. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The kinase promotes the transcriptional function of the tumor protein T. A. H. H. Y. W. H. M. Cell Biol. 2002; PubMed Scopus Google Scholar, G. B. T. I. Y. S. M. S. A. Del Sal G. G. M. E. S. Cell Biol. 2002; PubMed Scopus Google Scholar). at low levels in most normal Mirk is expressed at levels in skeletal and muscle (13Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar, Friedman E. Cancer Res. Scholar). The role of Mirk in muscle differentiation is to be of Mirk as a transcriptional as as more of Mirk as a Mirk has been shown to in a as a of the factor hepatocyte nuclear factor (11Lim S. Jin K. Friedman E. J. Biol. Chem. 2002; 277: 25040-25046Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Mirk to through its which as a to the binding of the to We that Mirk may as a of of the for the We also Y. S. Lee K. Deng X. Friedman E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) that Mirk functions to cell study function of Mirk/dyrk1B, phosphorylation of the CDK inhibitor p27 at Ser-10. p27 as a the Mirk phosphorylation of p27 may myoblasts to in G0 by stabilizing p27.