Mirk Regulates the Exit of Colon Cancer Cells from Quiescence

Mirk/Dyrk1B is a serine/threonine kinase widely expressed in colon cancers. Serum starvation induced HD6 colon carcinoma cells to enter a quiescent G0 state, characterized by a 2N DNA content and a lower RNA content than G1 cells. Compared with cycling cells, quiescent cells exhibited 16-fold higher levels of the retinoblastoma protein p130/Rb2, which sequesters E2F4 to block entry into G1, 10-fold elevated levels of the CDK inhibitor p27kip1, and 10-fold higher levels of Mirk. However, depletion of Mirk did not prevent entry into G0, but enabled quiescent HD6, SW480, and colo320 colon carcinoma cells to acquire some biochemical characteristics of G1 cells, including increased levels of cyclin D1 and cyclin D3 because of slower turnover, increased activity of their CDK4/cyclin D complexes, and increased phosphorylation and decreased E2F4 sequestering ability of the CDK4 target, p130/Rb2. As a result, depletion of Mirk allowed some cells to escape quiescence and enabled cells released from quiescence to traverse G1 more quickly. The kinase activity of Mirk was increased by the chemotherapeutic drug 5-fluorouracil (5-FU). Treatment of p53 mutant colon cancer cells with 5-FU led to an elongated G1 in a Mirk-dependent manner, as G1 was shortened by ectopic overexpression of cyclin D1 mutated at the Mirk phosphorylation site (T288A), but not by wild-type cyclin D1. Mirk, through regulating cyclin D turnover, and the CDK inhibitor p27, as shown by depletion studies, functioned independently and additively to regulate the exit of tumor cells from quiescence. Mirk/Dyrk1B is a serine/threonine kinase widely expressed in colon cancers. Serum starvation induced HD6 colon carcinoma cells to enter a quiescent G0 state, characterized by a 2N DNA content and a lower RNA content than G1 cells. Compared with cycling cells, quiescent cells exhibited 16-fold higher levels of the retinoblastoma protein p130/Rb2, which sequesters E2F4 to block entry into G1, 10-fold elevated levels of the CDK inhibitor p27kip1, and 10-fold higher levels of Mirk. However, depletion of Mirk did not prevent entry into G0, but enabled quiescent HD6, SW480, and colo320 colon carcinoma cells to acquire some biochemical characteristics of G1 cells, including increased levels of cyclin D1 and cyclin D3 because of slower turnover, increased activity of their CDK4/cyclin D complexes, and increased phosphorylation and decreased E2F4 sequestering ability of the CDK4 target, p130/Rb2. As a result, depletion of Mirk allowed some cells to escape quiescence and enabled cells released from quiescence to traverse G1 more quickly. The kinase activity of Mirk was increased by the chemotherapeutic drug 5-fluorouracil (5-FU). Treatment of p53 mutant colon cancer cells with 5-FU led to an elongated G1 in a Mirk-dependent manner, as G1 was shortened by ectopic overexpression of cyclin D1 mutated at the Mirk phosphorylation site (T288A), but not by wild-type cyclin D1. Mirk, through regulating cyclin D turnover, and the CDK inhibitor p27, as shown by depletion studies, functioned independently and additively to regulate the exit of tumor cells from quiescence. Solitary disseminated tumor cells that are negative for proliferation markers such as Ki67 are thought to be the source of tumor recurrence. These dormant tumor cells are quiescent, reversibly arrested in the G0 part of the cell cycle, and can re-enter the cell cycle under favorable clues from the microenvironment. Quiescence is not simply a long G1 period, but is characterized by specific changes in gene expression (1Coller H. Sang L. Roberts J.M. PLOS Biol. 2006; 4: 329-349Crossref Scopus (352) Google Scholar). Factors that allow the prolonged survival of quiescent tumor cells in vivo are of clinical relevance and include antioxidant proteins. A requirement for quiescence in normal hematopoietic stem cells is repression of the production of reactive oxygen species (ROS) 2The abbreviations used are: ROSreactive oxygen speciesRNAiRNA interference5-FU5-fluorouracilCDKcyclin-dependent kinaseFBSfetal bovine serum. (2Chen C. Liu Y. Liu R. Ikenoue T. Guan K.L. Liu Y. Zheng P. J. Exp. Med. 2008; 205: 2397-2408Crossref PubMed Scopus (553) Google Scholar), making it likely that cancer cells also require low ROS levels to maintain quiescence. The serine/threonine kinase Mirk was recently shown to reduce ROS levels in two pancreatic cancer cell lines by increasing transcription of a cohort of genes that detoxify superoxides and prevent the generation of hydroxyl radicals (3Deng X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar). The ROS-countering activity of Mirk was primarily exhibited in quiescent tumor cells because such cells had the highest levels of Mirk protein. Thus Mirk maintains the viability of quiescent pancreatic cancer cells through up-regulating antioxidant genes. reactive oxygen species RNA interference 5-fluorouracil cyclin-dependent kinase fetal bovine serum. Mirk/Dyrk1B is a member of a conserved family of serine/threonine kinases that are activated by intramolecular tyrosine phosphorylation and which mediate maturation in different tissues: Mirk in skeletal muscle, Dyrk1A in the brain, etc. One role of Mirk in skeletal muscle differentiation after a stress signal of serum deprivation is to block cycling myoblasts in the G0 quiescent state (4Deng X. Ewton D.Z. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) by phosphorylation of the cell cycle regulators cyclin D1 and p27kip1 (5Zou Y. Ewton D.Z. Deng X. Mercer S.E. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 6Deng X. Mercer S.E. Shah S. Ewton D.Z. Friedman E. J. Biol. Chem. 2004; 279: 22498-22504Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Phosphorylation by Mirk at a conserved ubiquitination site initiated proteolysis of cyclin D1, while p27kip1 was stabilized following phosphorylation by Mirk at Ser-10. Recently the survival factors Bcl2 and BclXL were shown to mediate G0 quiescence in NIH3T3 cells and murine embryonic fibroblasts through increasing p27kip1 stability, which required phosphorylation of p27Ser-10 by Mirk (7Janumyan Y. Cui Q. Yan L. Sansam C.G. Valentin M. Yang E. J. Biol. Chem. 2008; 283: 34108-34120Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These observations are consistent with Mirk function as a G0 kinase in nontransformed cells. Recently, observations have pointed to the importance of cancer cells arrested in a reversible quiescent state to undergo repair. Cell cycle restriction through the CDK inhibitor p21cip1 was shown to both limit DNA damage and maintain the self-renewal of leukemia stem cells (8Viale A. De Franco F. Orleth A. Cambiaghi V. Giuliani V. Bossi D. Ronchini C. Ronzoni S. Muradore I. Monestiroli S. Gobbi A. Alcalay M. Minucci S. 2009; PubMed Scopus Google Scholar). of in hematopoietic stem cells induced DNA damage and activated a that led to reversible cell cycle in a quiescent state and DNA repair. The that include of p21cip1 in leukemia stem cells (8Viale A. De Franco F. Orleth A. Cambiaghi V. Giuliani V. Bossi D. Ronchini C. Ronzoni S. Muradore I. Monestiroli S. Gobbi A. Alcalay M. Minucci S. 2009; PubMed Scopus Google Scholar). Mirk the to function as a G0 kinase to cell cycling in cancer cells. function of Mirk is the of the D1, and and to were from to a at the of Mirk was as Deng X. Friedman E. Cancer Google Scholar). and were from cell lines and the were from and in S. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The HD6 of the was M. M. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Deng X. M. Y. M. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Deng X. Friedman E. Cancer Google Scholar), is mutant in but wild-type genes F. S. Yan S. Friedman E. Google Scholar). The wild-type cyclin D1 and cyclin had in the in (5Zou Y. Ewton D.Z. Deng X. Mercer S.E. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). were at in cells were of Mirk for to quiescence. was as after with for following with and (3Deng X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar). different of the Mirk/Dyrk1B initiated at were from and are to Mirk by to p27kip1 was also from were in were with in for of cell of was with of at the were by the of of protein for at with and by of E2F4 and was as (3Deng X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar). The kinase activity of Mirk was as S. Mercer S.E. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with the protein from with as X. Ewton D.Z. Mercer S.E. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). CDK kinase activity was CDK4 to in an in kinase retinoblastoma protein was as S. Mercer S.E. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HD6 colon carcinoma cells were for after to cell and into the cell the of X. S. Yan Friedman E. Cell Google Scholar), that cells can enter a quiescent Mirk cell cell lines were used in which the Mirk gene was Mirk protein levels higher than in cancer cell lines (3Deng X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar). the Mirk gene is not in HD6 cells, which be more of the cancer cell in which Mirk is Mirk in cell colon cancer cells were for the of Mirk function Deng X. Friedman E. Cancer Google Scholar). that HD6 cells be to reversibly enter a quiescent state the cells were for in in A used to to DNA by to The of cells had a 2N DNA content by after and in cells that had with a 2N DNA content also had a low RNA were in G0 of the cells were in to of cells released by a to the chemotherapeutic drug 5-FU to and the inhibitor to block cycling cells in cell cycle was allowed in the of by that of cells were in G0, with cells in cell cycle while after from cells had the cell cycle, with in G0 Thus the of the quiescent HD6 cells of the cell cycle under depletion colon cancer cells to traverse G1 released from but not cycling of HD6 colon carcinoma cells were of Mirk by and quiescent as in were released by to and the inhibitor at cell cycle be by after shown are of with the shown in Mirk depletion not cell cycle of cells were of Mirk by with and with by a in with in by in were released by a to and to cycling cells in and were for cell cycle by and following The of cells was with in from the shown in A and Mirk and levels in HD6 colon carcinoma cells and in fibroblasts depletion the cycling of colon cancer cells, but not of normal fibroblasts as by in HD6 colon carcinoma in in in in a A was used to the quiescent Mirk protein levels increased as did levels of the CDK inhibitor p27kip1, a of quiescent cells The retinoblastoma protein family member sequesters the E2F4 transcription of cells into The protein of is elevated in quiescent cells and decreased in cells Cell Google Scholar). HD6 cells exhibited 16-fold higher levels of than cells in cells are to enter G1 by of is by G1 and more HD6 cells were in their exhibited while cells a quiescent state after of serum their species exhibited a more consistent with phosphorylation and lower The were more in a lower cells are to enter G1 by of is by G1 complexes, more and of Mirk the as of of by phosphorylation into species and Treatment of with of the into in a Thus Mirk depletion in quiescent colon carcinoma cells increased the activity of the CDK4/cyclin D which the protein by quiescent HD6 cells in G0 the G1 biochemical of of Mirk. The role of Mirk in quiescent cells was by RNA interference of cells a G0 quiescent state, but cells were in G0 and more in and not of Mirk in quiescent pancreatic cancer cells led to increased cell because of a higher of ROS (3Deng X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar). A in ROS levels was in colon cancer cells not that the of cells from quiescent colon cancer was a Mirk was quiescent cells were released into more cells cycle, with in and for the that cells were more of the quiescent Mirk was by two to a different of the Mirk and were after more HD6, and colon cancer cells than cells had from quiescence serum starvation and had a DNA content in and of from the of HD6 cells of Mirk by were in while the of the cells in were cells were released into of two of Mirk quiescent colon cancer cells enter cycle serum factors were Mirk is widely expressed in normal at low levels Deng X. Friedman E. Cancer Google Scholar), depletion of Mirk allow quiescent normal cells to re-enter of the of normal fibroblasts in led cells to with a 2N DNA content of Mirk by of two with and A of and cells were in and after from and not Thus depletion of Mirk had the cycling of normal because cells had a normal G1 of the colon cancer cell lines in and had mutant the G1 Mirk protein was not in colon cancer cell lines and with wild-type p53 not the of Mirk depletion not be in depletion the cycling of colon cancer cells but not of normal fibroblasts as by in in in in in a depletion of Mirk did not expression of cell cycle genes such as CDK However, in with nontransformed cells overexpression of Mirk was to a reversible in G0 because Mirk the G1 cyclin D1, for by phosphorylation at a ubiquitination site conserved in cyclin D (5Zou Y. Ewton D.Z. Deng X. Mercer S.E. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). levels of cyclin D1 are in colon cancer J. R. D. M. J. J. PubMed Scopus Google Scholar) cells were to some of of Mirk in and colo320 colon cancer cells by two to different of the Mirk led to and in cyclin D1 because of an in cyclin D not quiescent HD6 cells had an of more cyclin D3 and cyclin D1 Mirk had because of and and not The Mirk phosphorylation site is conserved D1 and and Mirk shown to the of both in pancreatic cancer cells (3Deng X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar). of Mirk in HD6 cells increased the of cyclin D1 with cells for the increased protein studies, Mirk of colon cancer cells were shown to a of cyclin D1 D.Z. Deng X. S. Friedman E. J. 2003; PubMed Scopus Google Scholar). both Mirk depletion and Mirk overexpression that Mirk cyclin D in colon cancer cells. from G0 into G1 and through G1 is by CDK4/cyclin complexes, which of the retinoblastoma protein and p130/Rb2. of Mirk led to more phosphorylation of consistent with increased CDK4 CDK4 activity was by of CDK4/cyclin from and quiescent of HD6 cells, and for from after from quiescence levels of D1, and were at low levels as cells had cycle and quiescent cells exhibited as CDK4 activity as quiescent HD6 cells, consistent with their higher levels of cyclin D3 and D1 HD6 cells were through and after from quiescence their CDK4 activity was the low levels of G1 and of Mirk led to of the G0 protein p130/Rb2, the retinoblastoma protein family member that entry into Mirk was in HD6 and in cells by a different than that used in but in both decreased of was Mirk is consistent with more phosphorylation by cyclin were The were by by for E2F4 with with by the of in the was E2F4 with in HD6 cells and in cells Thus depletion of Mirk increased the of in more with lower that have ability to the transcription E2F4 which entry into As a result, quiescent HD6 cells, primarily in G0 some biochemical markers of cells in elevated levels of G1 elevated CDK4 kinase activity the retinoblastoma protein and and DNA damage in tumor cells with mutant p53 Cancer Full Text Full Text PDF PubMed Scopus Google Scholar), such as HD6, SW480, and colo320 colon cancer cells. The kinase Mirk is activated by the kinases and S. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Y. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Mercer S.E. Friedman E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which J. T. B. Biol. PubMed Scopus Google Scholar), that stress be to Mirk. the 5-FU induced a in Mirk kinase activity with at for Treatment with 5-FU led to lower cyclin D1 levels by the of cyclin D1 through of Mirk, that 5-FU of Mirk cell colon cancer cell lines were quiescent with at of cells in released into 5-FU and the inhibitor to block cycling cells in cells have wild-type p53 released from were arrested by 5-FU at a G1 through p53 of p21cip1 while cells not with 5-FU through G1 and and were in by SW480, and HD6 cells are in p53 G1 released from quiescence into 5-FU for some cells from of lines were in G1 and not as as at the and to be by the and kinases J. 2003; PubMed Scopus Google Scholar, R. M. R. J. 2006; PubMed Scopus Google Scholar, J. H. Cancer 2006; PubMed Scopus Google Scholar). DNA damage initiated by 5-FU to a G1 in some tumor cells by R. M. H. M. T. T. Cancer Google Scholar), of the in G1 of HD6 and colo320 not colon cancer cells. more Mirk mediate some of G1 in p53 mutant colon cancer cells, were following Mirk HD6 of cells were quiescent, released for in the of and cell cycle was were released in the of the inhibitor cell cycle was of the cells were in G1 A and while in the cells primarily in and at activated by As by after of of the cells were in G1, while of the cells had G1 and were in Thus 5-FU not G1 in the cells, which prevent their entry into 5-FU increased the of G1 in HD6 cells because that 5-FU was not quiescent and the G1 than in G1 after were by with 5-FU induced a G1 in HD6 cells Mirk was and the G1 was Mirk was not expression of G1 was to DNA in quiescent fibroblasts L. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) while overexpression of shortened the G1 Roberts J.M. 2004; PubMed Scopus Google Scholar), cyclin D cycling of HD6 cells. the phosphorylation of cyclin D was the of Mirk in through G1, overexpression of a cyclin D1 mutated at the Mirk phosphorylation site block Mirk function and allow cells to cycle into be by wild-type cyclin D1 and the mutant cyclin had in the in (5Zou Y. Ewton D.Z. Deng X. Mercer S.E. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and were expressed at a as shown by with to cyclin D1 an to their The cyclin D1 were as as the cyclin D1 were likely to the in with G1 HD6 cells were with cyclin wild-type cyclin D1, quiescent, released for into 5-FU and a inhibitor cell cycle was the with ectopic wild-type cyclin D1 of the cells were in G1, while in the Mirk site mutant cyclin cells G1 and 5-FU activated the with of cells arrested A and is a of the of Mirk depletion cell cycle in of the cells were in G1, and of the cells were in S. the cell cycle function of Mirk in colon cancer cells is the cyclin D that through overexpression of mutant cyclin with Mirk depletion led to more through the cell cycle with the of cells in not the of of cells with wild-type cyclin D1 and of cells with the mutant cyclin D1 were in and than in G1, under the in not with cyclin D1 of of cells had to and were arrested by not Thus the wild-type the mutant cyclin D cells in G1 under Mirk cycling myoblasts in the G0 quiescent state (4Deng X. Ewton D.Z. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), in part by of the p27kip1 protein by phosphorylation at X. Mercer S.E. Shah S. Ewton D.Z. Friedman E. J. Biol. Chem. 2004; 279: 22498-22504Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). the survival factors Bcl2 and BclXL were shown to mediate G0 quiescence in NIH3T3 cells and murine embryonic fibroblasts through increasing p27kip1 stability, in a which required phosphorylation of by Mirk (7Janumyan Y. Cui Q. Yan L. Sansam C.G. Valentin M. Yang E. J. Biol. Chem. 2008; 283: 34108-34120Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These that Mirk elevated levels to nontransformed cells in a quiescent Mirk the exit of HD6 colon carcinoma cells from G0 by as as cyclin Mirk and independently to maintain quiescence. HD6 cells were of Mirk, both Mirk and Mirk depletion did not levels in and also not that Mirk did not function to in cancer cells. were quiescent and released by a to 5-FU and to prevent the released cells from more than cell The were that of the released cells were in G0 and were in G1 of Mirk led cells to more from G0 into G1 with a of enabled cells to both G0 and G1, of cells to enter depletion of Mirk and independently the of cells in G0 to and depletion of Mirk and had an the of G0 cells from to and of the cells to into and and Mirk the exit of HD6 colon carcinoma cells from G0 by in an cells their G0 cells to be by their 2N DNA content and their low RNA content and by the of biochemical including a to and sequestering the transcription E2F4 and elevated levels of the CDK inhibitor cells arrested in G0 by serum are by a L. S. L. X. S. R. Liu Full Text Full Text PDF PubMed Scopus Google Scholar). normal in G0 is an by a of from in cell cycle The transcription is a survival for quiescent but not carcinoma cells and 2008; PubMed Scopus Google Scholar). Recently was shown to maintain quiescence and function of hematopoietic stem cells by and the generation of reactive oxygen species (2Chen C. Liu Y. Liu R. Ikenoue T. Guan K.L. Liu Y. Zheng P. J. Exp. Med. 2008; 205: 2397-2408Crossref PubMed Scopus (553) Google Scholar). The quiescence of stem cells is for their long function and is thought to require the in their cells have and can which is to be to hematopoietic stem cells. of in a led to more more and the of quiescent stem cells into cycling (2Chen C. Liu Y. Liu R. Ikenoue T. Guan K.L. Liu Y. Zheng P. J. Exp. Med. 2008; 205: 2397-2408Crossref PubMed Scopus (553) Google Scholar). cells are also in DNA 2004; PubMed Scopus Google Scholar), and as a result, are to by DNA fibroblasts the gene in a have used to DNA damage and in quiescent and in cells in of such quiescent normal fibroblasts led to damage in the and as with cells, and quiescent cells were in PubMed Scopus Google Scholar, DNA 2004; PubMed Scopus Google Scholar). The to enter quiescence is of the characteristics of stem cells in S. 2004; PubMed Scopus Google Scholar, T. H. Yang Y. D. M. PubMed Scopus Google Scholar, D. PubMed Scopus Google Scholar). quiescent stem cells have that are to Cell 2006; PubMed Scopus Google Scholar), because of their increased for repair. stem cells required a in a quiescent state to DNA damage induced by of (8Viale A. De Franco F. Orleth A. Cambiaghi V. Giuliani V. Bossi D. Ronchini C. Ronzoni S. Muradore I. Monestiroli S. Gobbi A. Alcalay M. Minucci S. 2009; PubMed Scopus Google Scholar). requirement for quiescence is not to leukemia cells. colon a G0 quiescence state were more to stress damage I. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). the the kinase Mirk shown to prevent colon carcinoma cells from a quiescent G0 state, at in through of G1 Mirk was recently shown to reduce ROS levels in two pancreatic cancer cell lines by increasing transcription of a cohort of genes which detoxify superoxides and which prevent the generation of hydroxyl radicals (3Deng X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar). The ROS-countering activity of Mirk was primarily exhibited in quiescent tumor cells, because such cells had the highest levels of Mirk protein. colon cancer cells Mirk was at highest in G0 it also ROS levels not and cell cycle colon cancer cell HD6, SW480, and colo320 of Mirk by of two were to G1 including cyclin D1, had quiescent by serum levels of G1 activated the CDK4 kinase which in increased phosphorylation of retinoblastoma proteins. These the protein that entry into G1 by sequestering the transcription As a result, depletion of Mirk led to increased phosphorylation of p130/Rb2, which sequestering for Thus quiescent colon cancer cells of Mirk some of the of cells in of quiescence by of serum factors led to a more of G1 than that in cells, as cells were to of a quiescent cells G0 released from quiescence and in G1, while depletion of both Mirk and the CDK inhibitor enabled cells to exit G0 and enter cycle a colon cancer cells of G0, G1, and were in in the of serum factors and depletion of Mirk the cycling of colon cancer cells, the of of Mirk were 5-FU activated Mirk as a kinase and 5-FU allowed HD6 cells to undergo a prolonged G1 in a Mirk-dependent One of the Mirk in a of cell is the cyclin D of G1 which Mirk at a conserved ubiquitination in cyclin D1 (5Zou Y. Ewton D.Z. Deng X. Mercer S.E. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), F. J. E. Y. M. Y. S. T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The role of phosphorylation of cyclin D was by the cell cycling of cyclin mutated at the Mirk phosphorylation of mutant cyclin D Mirk from a G1 by of cyclin D. The G1 was ectopic wild-type cyclin D1 was expressed that Mirk phosphorylation of cyclin D1 was an part of Mirk function in the is widely for of cyclin D1 by phosphorylation at Cancer Biol. PubMed Scopus Google Scholar). Mirk cyclin D1 at the site of in a of cancer cells and nontransformed cells (5Zou Y. Ewton D.Z. Deng X. Mercer S.E. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, F. J. E. Y. M. Y. S. T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, X. Ewton D.Z. Friedman E. Cancer Research. 2009; 69: 3317-3324Crossref PubMed Scopus (70) Google Scholar), with both to be following phosphorylation D. A. A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Mirk was to cyclin D1 to while Mirk and cyclin D1 in (5Zou Y. Ewton D.Z. Deng X. Mercer S.E. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The kinase Dyrk1A to as a kinase for in phosphorylation of P. R. M. X. C.G. J. PubMed Scopus Google Scholar), and in some cells as a for of cyclin D by The protein as a to of an S. J. Cell Biol. 2009; PubMed Scopus Google Scholar), that family of kinases in protein