The Rb-CDK4/6 Signaling Pathway Is Critical in Neural Precursor Cell Cycle Regulation

The tumor suppressor, retinoblastoma (Rb), is involved in both terminal mitosis and neuronal differentiation. We hypothesized that activation of the Rb pathway would induce cell cycle arrest in primary neural precursor cells, independent of the proposed function of cyclin-dependent kinases 4/6 (CDK4/6) to sequester the CIP/KIP CDK inhibitors (CKIs) p21 and p27 from CDK2. We expressed dominant negative adenovirus mutants of CDKs 2, 4, and 6 (dnCDK2, dnCDK4, and dnCDK6) in neural progenitor cells derived from E12.5 wild type and Rb-deficient mouse embryos. In contrast to previous studies, our results demonstrate that in addition to dnCDK2, the dnCDK4/6 mutants can induce growth arrest. Moreover, the dnCDK4/6-mediated inhibition is Rb-dependent. The dnCDK2 partially inhibited cell growth in Rb-deficient cells, suggesting that CDK2 may have additional targets. A previously proposed function of CDK4/6 is CKI sequestration, thereby preventing the resulting inhibition of CDK2, believed to be the key regulator of cell cycle. However, our immunoprecipitations revealed that the dominant negative CDK mutants could arrest cell growth despite their interaction with p21 and p27. Taken together, our results demonstrate that both CDK2 and CDK4/6 are crucial for cell cycle regulation. Furthermore, our data underscore the importance of the Rb regulatory pathway in neuronal development and cell cycle regulation, independent of CKI sequestration. The tumor suppressor, retinoblastoma (Rb), is involved in both terminal mitosis and neuronal differentiation. We hypothesized that activation of the Rb pathway would induce cell cycle arrest in primary neural precursor cells, independent of the proposed function of cyclin-dependent kinases 4/6 (CDK4/6) to sequester the CIP/KIP CDK inhibitors (CKIs) p21 and p27 from CDK2. We expressed dominant negative adenovirus mutants of CDKs 2, 4, and 6 (dnCDK2, dnCDK4, and dnCDK6) in neural progenitor cells derived from E12.5 wild type and Rb-deficient mouse embryos. In contrast to previous studies, our results demonstrate that in addition to dnCDK2, the dnCDK4/6 mutants can induce growth arrest. Moreover, the dnCDK4/6-mediated inhibition is Rb-dependent. The dnCDK2 partially inhibited cell growth in Rb-deficient cells, suggesting that CDK2 may have additional targets. A previously proposed function of CDK4/6 is CKI sequestration, thereby preventing the resulting inhibition of CDK2, believed to be the key regulator of cell cycle. However, our immunoprecipitations revealed that the dominant negative CDK mutants could arrest cell growth despite their interaction with p21 and p27. Taken together, our results demonstrate that both CDK2 and CDK4/6 are crucial for cell cycle regulation. Furthermore, our data underscore the importance of the Rb regulatory pathway in neuronal development and cell cycle regulation, independent of CKI sequestration. retinoblastoma cyclin-dependent kinase CDK inhibitor bromodeoxyuridine multiplicity of infection During embryogenesis, cycling neural progenitor cells in the ventricular zones commit to a neuronal fate and as a consequence of that decision undergo terminal mitosis and adopt a neuronal phenotype. A key developmental step in this process is the decision to undergo terminal mitosis. Several lines of evidence indicate that terminal mitosis and terminal differentiation are intimately coupled. First, cells in the mitotic layer of the developing retina induce expression of a retinal ganglion cell marker within minutes of the S phase of terminal mitosis (1McLoon S.C. Barnes R.B. J. Neurosci. 1989; 9: 1424-1432Crossref PubMed Google Scholar, 2Waid D.K. McLoon S.C. Neuron. 1995; 14: 117-124Abstract Full Text PDF PubMed Scopus (128) Google Scholar). Second, neural precursor cells transplanted throughout the developing cortex exhibit appropriate neuronal identify only if they had undergone terminal mitosis following implantation (3McConnell S.K. Kaznowski C.E. Science. 1991; 254: 282-285Crossref PubMed Scopus (622) Google Scholar). Third, sensory neurons override the G1 restriction point and undergo S-phase death in the absence of the neurotrophin, NT-3 (4ElShamy W.M. Fridvall L.K. Ernfors P. Neuron. 1998; 21: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The importance of terminal mitosis in neuronal development is underscored by the fact that failure to permanently withdraw from the cell cycle results in impaired differentiation and apoptosis. One key regulator of the cell cycle, the tumor suppressor protein pRb, has recently been implicated in terminal mitosis and neuronal differentiation. Mice nullizygous for Rb1 die by E15.5 from hematopoietic and neurological defects attributed to failed terminal differentiation (5Clarke A.R. Maandag E.R. Vam Roon M. van der Lugt N.M.T. van der Valk M. Hooper M.I. Berns A. Te Reile H. Nature. 1992; 359: 328-330Crossref PubMed Scopus (891) Google Scholar, 6Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1514) Google Scholar, 7Lee E.H.P. Chang C.Y. Hu N. Wang Y.C.J. Lai C.C. Herrup K. Lee W.H. Bradley A. Nature. 1992; 359: 288-294Crossref PubMed Scopus (1119) Google Scholar). By E12.5 onward, ectopic mitoses and massive cell death are observed throughout the developing nervous system. Studies examining neuronal development have revealed reduced expression of pan-neuronal genes as well as the nerve growth factor receptor TrkA, suggesting that Rb has an important role in differentiation (8Lee E. Hu N. Yuan S.S.F. Cox L.A. Bradley A. Lee W. Herrup K. Genes Dev. 1994; 8: 2008-2021Crossref PubMed Scopus (269) Google Scholar). Examination of the expression of a neuron-specific transgene in Rb-deficient embryos revealed impaired neurogenesis in virtually all neuronal cell types examined (9Slack R.S. El-Bizri H. Wong J. Belliveau D.J. Miller F.D. J. Cell Biol. 1998; 140: 1497-1509Crossref PubMed Scopus (87) Google Scholar). Furthermore, Rb-deficient neural precursor cells exhibited a dramatic delay in their ability to undergo terminal mitosis (10Callaghan D.A. Dong L. Callaghan S.M. Hou Y.X. Dagnino L. Slack R.S. Dev. Biol. 1999; 207: 257-270Crossref PubMed Scopus (63) Google Scholar), indicating that Rb plays an important role in regulating the onset of terminal mitosis in progenitor cells. While Rb is thought to be a key regulator of terminal mitosis in neural precursor cells (reviewed in Ref. 11Slack R.S. Miller F.D. Dev. Genet. 1996; 18: 81-91Crossref PubMed Scopus (26) Google Scholar), there is little evidence that activation of the Rb pathway is sufficient to induce cell cycle arrest in this cell type. Furthermore, although it is well established that CDK2, CDK4, and CDK6 are all involved in cell cycle regulation, their precise roles and importance are still the subject of controversy. Indeed, previous studies examining the role of cyclin-dependent kinases (CDKs) in cell cycle regulation have indicated that inhibition of CDK4/6, which function through the Rb pathway, is insufficient to induce mitotic arrest in several cell types. Specifically, a dominant negative mutant of CDK2, which sequesters cyclins A and E from endogenous CDKs, could efficiently induce growth arrest, while dominant negative mutants of CDK4/6 which sequester D cyclins, were unable to mediate cell cycle arrest (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google Scholar). One interpretation of these results is that activation of the Rb pathway alone may not be sufficient to mediate arrest of cell growth. It has recently been suggested that CDK4/6, in its association with D cyclins, may have dual functions. First, these proteins inactivate Rb activity by phosphorylation (13Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (875) Google Scholar, 14Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J.-Y. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 15Kato J.-Y Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1090) Google Scholar, 16Matsushime H. Quelle D.E. Shurtleff S.A. Masabumi S. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1025) Google Scholar, 17Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Crossref PubMed Scopus (735) Google Scholar, 18Mittnacht S. Lees J.A. Desai D. Harlow E. Morgan D.O. Weinberg R.A. EMBO J. 1994; 13: 118-127Crossref PubMed Scopus (126) Google Scholar). Second, CDK4/6 may sequester the CIP/KIP CDK inhibitors (CKIs) p21 and p27, thus rendering them unavailable to bind and inactivate CDK2 (19Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5130) Google Scholar). Recently, p21 and p27 have been shown to associate with CDK4/6 in a facilitatory CDK-cyclin interaction without inhibition of kinase activity (19Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5130) Google Scholar, 20LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. A. Harlow E. Genes Dev. PubMed Scopus Google Scholar, M. P. J.A. M. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: PubMed Scopus Google Scholar). In as the and they thereby them to associate with CDK2, an interaction G1 arrest (19Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5130) Google Scholar, J. Genes Dev. PubMed Scopus Google Scholar, K. A. J. Genes Dev. 1995; 9: PubMed Scopus Google Scholar). it is believed that the inhibition of E activity is the crucial step in cell cycle regulation. The of CDK2 as the regulator of cell cycle with CDK4/6 has (19Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5130) Google Scholar). While this is by a of studies, it be that the dominant negative mutants were tumor cells and may not the of cell cycle regulation in primary cells. In of the importance of as by the nervous defects in for hypothesized that activation of the Rb pathway alone would be sufficient to induce cell cycle arrest in primary neural precursor cells, independent of its function in the of the proteins from CDK2. this have primary neural progenitor cells derived from E12.5 wild type and Rb-deficient embryos. negative CDK mutants were these cells adenovirus In contrast to studies cell our results demonstrate that dominant negative mutants of and CDK6 can induce mitotic arrest in primary neural precursor cells, independent of the previously proposed and of p21 and p27. The fact that these CDK4/6 mutants are in cells that Rb that the activation of the Rb pathway in is sufficient to cell cycle arrest in primary neural precursor cells. Rb-deficient by T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1514) Google Scholar), were from and a Mice were by as previously T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1514) Google from for from for cell were and the of as of primary neural precursor cells, embryos were progenitor cells were as previously A. M.E. Neuron. 1995; Full Text PDF PubMed Scopus Google Scholar), with (9Slack R.S. El-Bizri H. Wong J. Belliveau D.J. Miller F.D. J. Cell Biol. 1998; 140: 1497-1509Crossref PubMed Scopus (87) Google Scholar). were from E12.5 mouse and were The of and In growth factor only with the with Rb-deficient progenitor cells, the from embryos and the appropriate wild type and were for neurons were from from which were and in with and a primary precursor cell neural cells were from the the from mouse embryos and to a cells were a of in growth factor as previously S. Neuron. 1993; Full Text PDF PubMed Scopus Google Scholar). cells were as in and were to a and cells were for and were not cells were in for to a in cells were for in and with cells were with the appropriate to to with cells were with for to and with for were for with and to an with the primary were with a for and for in to The for the dominant negative mutants of CDK2, CDK4, and CDK6 were from Harlow (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google and were with the L.A. J. Neurosci. PubMed Google Scholar). The CDK mutants were the of the S. M. T. J. PubMed Google that with the and in cells as previously S. M. T. J. PubMed Google Scholar). The the expression by in the of and progenitor cells were in with in of of the appropriate of the adenovirus to following a The multiplicity of infection the of cells were with adenovirus The appropriate and for 6 which a The cells were and the in were for protein in A for by a a and to a in were in the primary for in were for in the were by to the cells the dominant negative CDK mutants were in and for Cell of as well as a in were with The were in and by for in The for with the appropriate cyclins and as in the to and negative CDK mutants are with the inhibitors p21 and p27. cells were with following cells were and cell of and only in were with The were with p21 and p27 dnCDK2 and The following primary were mouse mouse and mouse mouse and mouse were from mouse mouse A and E to the role of the Rb pathway in cell cycle regulation of neural precursor cells, to the expression of the cyclin-dependent cyclins, and cyclin-dependent kinase inhibitors the of differentiation. cells, a and neurons of a were of as a for First, the expression of the CDKs were examined the differentiation of progenitor cells. the differentiation in CDK2 protein to and CDK6 and are expressed in neural precursor cells and protein as neurons In the expression of which is expressed in neuronal cells and not believed to function in the regulation of cell cycle H. T. T. M. 1994; PubMed Scopus Google Scholar, T. Harlow E. Nature. 1994; PubMed Scopus Google Scholar), is the of with the of neurons in these in progenitor cells, CDK expression is that the of CDK2, CDK4, and CDK6 to while is as neurons are We examined the expression of cyclins the of neurogenesis The D cyclins, cyclins and are all expressed neuronal differentiation in in their regulation were is in cells as cells undergo with expression in neurons In is expressed in cells although in neuronal is both in and cells a CDK2 is expressed throughout the of differentiation However, to the observed with its CDK2, is expressed in cells and as neurons are While CDK activity is several the expression of the as well as the expression of the kinase key roles in cell cycle regulation neural differentiation V. van der A. J. Cell Biol. 1995; PubMed Scopus Google Scholar, Slack M. P. Cell 1998; PubMed Scopus Google Scholar). We examined the expression of the CKI as well as the CIP/KIP kinase inhibitors p21 and p27 is expressed in neural precursor cells and is as cells undergo differentiation p27 a expression in neural precursor cells that as differentiation In p21 in neural cells, which the of differentiation to in neurons The of p21 the in neural cells, is with the that p21 has that of inhibition of cyclin-dependent kinase activity J. Garrett M.D. Stevenson L.F. Slingerland J.M. A. Harlow E. Genes Dev. PubMed Scopus Google Scholar, M. P. J.A. M. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: PubMed Scopus Google Scholar). indicated that Rb is in neural and as neurogenesis of retinoblastoma protein progenitor cells were from wild type E12.5 embryos and to in neural cells and neurons for were for and protein and were with which CDKs are important in the regulation of cell cycle in primary neural precursor cells, adenovirus dominant negative mutants of CDK2, CDK4, and CDK6 (dnCDK2, dnCDK4, and dominant mutants have been previously (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google and a point in the kinase thereby rendering them mutants the ability to bind cyclins, thus they inactivate the function of endogenous CDKs by their that adenovirus could the CDK mutant neural progenitor cells were and these infection the of function and would be J. P. Slack R.S. 7: PubMed Scopus Google Scholar). for CDK2, CDK4, and CDK6 expression of the appropriate CDK mutants in progenitor cells expression by with an studies have shown that the dominant mutant of CDK2 of growth arrest in several cell mutants of CDK4/6 were (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google Scholar). these studies were in tumor cell the of CDK4/6, which function through the Rb pathway, may be important in cell cycle regulation in primary neural progenitor cells. type neural precursor cells were in and in cells were with to identify cells by with The of cells that were were and the of expressed as a of cells. The 6 In contrast to cells, which a of 6 there little in cells dnCDK2 6 of these results indicated that in in dnCDK2 cells, only were with previous studies (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google Scholar), our results indicate that dnCDK2 can efficiently induce growth arrest in progenitor cells. We and could induce growth arrest in neural precursor cells. Examination of progenitor cells revealed that the of cells were growth and not S phase Cell revealed that to cells and exhibited only and 6 results indicate that dominant negative which the activity of CDK4/6, are sufficient to induce growth arrest in neural precursor cells. While inhibition in previous studies cell our results that this is an important growth regulatory pathway and that CDK4/6 activation is for cell cycle in primary neural progenitor cells. studies have shown that cell cycle regulation by CDK4/6 through Rb phosphorylation H. Quelle D.E. Shurtleff S.A. Masabumi S. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1025) Google Scholar, 17Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Crossref PubMed Scopus (735) Google Scholar, 18Mittnacht S. Lees J.A. Desai D. Harlow E. Morgan D.O. Weinberg R.A. EMBO J. 1994; 13: 118-127Crossref PubMed Scopus (126) Google Scholar, J. J. M. M. J. Mol. Cell. Biol. 1995; PubMed Scopus Google of mutants in Rb cells have a to that the dnCDK4/6 mutants are the in Rb-deficient progenitor cells In the absence of cells a of to in wild type cells dnCDK2 to a in despite the absence of Rb of the indicated that dnCDK2 in the growth of Rb-deficient cells The ability of dnCDK2 to to that in neural precursor cells, CDK2 function is partially there are targets. with Rb as the for CDK4/6, the dominant negative mutants had cell growth cells exhibited and cells and results indicate that adenovirus the mutants are in neural precursor cells. In of the of the dominant CDK mutants in progenitor cell cycle regulation, to that the CDK mutants were as and thereby the that may to of The mutants were expressed in neural cells to their cells for they of and can be with adenovirus that of cells the were with dnCDK2, dnCDK4, and were for with to the mutant CDK The were examined for proteins by The were for a for CDK4/6 and cyclins A and which bind and all of which are expressed in neural cells 2, in from cells not in cells dnCDK2 cyclins A and E were with their appropriate the dnCDK2 not dnCDK4/6 results demonstrate that despite their the dominant negative mutants with their appropriate in neural precursor cells. it is that CDK4/6 function through the regulation of Rb activity by recently studies that these proteins have an additional function in the of the of have been shown to have CDK4/6 activity function to the CDK-cyclin (19Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5130) Google Scholar, 20LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. A. Harlow E. Genes Dev. PubMed Scopus Google Scholar, M. P. J.A. M. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: PubMed Scopus Google Scholar). One for the cell cycle inhibition of CDK4/6 is that they are to bind p21 p27 thereby rendering them to the activity of CDK2. We examined not the CDK mutants could with the CIP/KIP p21 and p27. that p21 in with dnCDK2, in dnCDK4/6 with previous K. Lee H. A. Roberts J.M. P. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, H. T. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), p27 in dnCDK4/6 results indicate that cell cycle arrest not by the of these kinase studies that can efficiently cell cycle despite their interaction with p21 and p27 and that cell cycle arrest in primary neural precursor cells through the regulation of Rb The onset of pan-neuronal expression is to terminal mitosis (1McLoon S.C. Barnes R.B. J. Neurosci. 1989; 9: 1424-1432Crossref PubMed Google Scholar, 2Waid D.K. McLoon S.C. Neuron. 1995; 14: 117-124Abstract Full Text PDF PubMed Scopus (128) Google Scholar, S.K. Kaznowski C.E. Science. 1991; 254: 282-285Crossref PubMed Scopus (622) Google Scholar, W.M. Fridvall L.K. Ernfors P. Neuron. 1998; 21: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). the that cell cycle is key to the While studies with Rb-deficient have the importance of this tumor suppressor in nervous development (5Clarke A.R. Maandag E.R. Vam Roon M. van der Lugt N.M.T. van der Valk M. Hooper M.I. Berns A. Te Reile H. Nature. 1992; 359: 328-330Crossref PubMed Scopus (891) Google Scholar, 6Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1514) Google Scholar, 7Lee E.H.P. Chang C.Y. Hu N. Wang Y.C.J. Lai C.C. Herrup K. Lee W.H. Bradley A. Nature. 1992; 359: 288-294Crossref PubMed Scopus (1119) Google Scholar), there is little evidence that Rb its can induce cell cycle arrest. In the demonstrate that the Rb pathway is a regulator of cell cycle in primary neural progenitor cells. In contrast to previous studies (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google Scholar), that dominant negative mutants of and CDK6 can induce mitotic arrest in neural precursor cells, despite the and of the p21 and p27. The activity of these mutants is the of indicating that the activation of the Rb pathway is sufficient to cell cycle arrest in primary neural precursor cells. The restriction point of the cell cycle is as the in G1 which cells growth to S phase Weinberg R.A. Cell Biol. 9: PubMed Scopus Google Scholar). Studies indicate that CDK2 and CDK4/6 function regulatory the G1 phase of the cell cycle. CDK4/6, with D cyclins, function through the of Rb in G1 to the restriction point (13Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (875) Google Scholar, 14Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J.-Y. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 15Kato J.-Y Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1090) Google Scholar, 17Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Crossref PubMed Scopus (735) Google Scholar). The through the restriction point and the of D cyclins is and the cell to S phase H. R.A. Sherr C.J. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, A. S. J. Cell 1993; PubMed Google Scholar). CDK2, with cyclins A and the cell cycle in which the cell is to undergo the phase V. Lees E. Reed S.I. Science. 1992; PubMed Scopus Google Scholar, M. J. Roberts J.M. M. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). studies have shown that dominant negative mutants of CDK2, not CDK4/6, in cell cycle arrest in several cell lines (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google Scholar). results are with the interpretation that CDK2 activity may be crucial for phase while CDK4/6 may a important role in regulating it has been suggested that CDKs 4/6 may have dual the regulation of Rb phosphorylation as well as the of p21 and p27 to the inhibition of CDK2 It is believed that this function may be a crucial in the regulation of the (19Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5130) Google Scholar, 20LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. A. Harlow E. Genes Dev. PubMed Scopus Google Scholar, M. P. J.A. M. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: PubMed Scopus Google Scholar). which CDKs are important cell cycle in neural precursor cells, dominant negative CDK mutants adenovirus of the dnCDK2 mutant kinase results in a growth arrest in neural precursor cells, by a in to previous results with cell lines (12van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (972) Google Scholar), dnCDK4/6 were cell cycle as cells these mutants exhibited a in results indicate that in addition to the established role for CDK2 in regulating cell cycle CDK4/6 activity is for neural precursor cells to S that mutants expressed dnCDK4/6 in Rb-deficient progenitor cells, and as our results that the cell growth regulation by CDK4/6 activity the Rb In the expression of dnCDK2 in Rb progenitor cells could a in cells. not to be as in the absence of suggesting that CDK2 partially through the Rb pathway as well as additional targets. It is that growth arrest is to resulting from adenovirus cells exhibited a in progenitor cells in (10Callaghan D.A. Dong L. Callaghan S.M. Hou Y.X. Dagnino L. Slack R.S. Dev. Biol. 1999; 207: 257-270Crossref PubMed Scopus (63) Google Scholar), and of dnCDK4/6 to Rb-deficient precursor cells had of the of In the and would the of J. P. Slack R.S. 7: PubMed Scopus Google Scholar). By examining the of the CDK have that the CDK mutants function in the with to their to their results that CDK4/6 activity is for cell cycle in primary neural precursor cells. studies of primary have growth arrest in an J. J. M. M. J. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). with our this that CDK4/6 regulation is for cell cycle in primary cells. It has been that may have the of the CIP/KIP thereby to CDK2 which the key in cell growth arrest. CDK4/6 have been shown to with the CIP/KIP of p21 and p27, for growth arrest is the of p21 and p27, rendering them to CDK2. the observed growth arrest could from the inhibition of CDK2 this in our studies, immunoprecipitations of the mutants to the ability to sequester these is results demonstrate that both p27 and p21 are with dnCDK4/6 that the dnCDK4/6 mutants the ability to bind the CIP/KIP and can arrest cell growth despite this inhibition of the CDK4/6 kinase activity is for S phase and Rb is the for this The results of the demonstrate that CDK4/6 their ability to is for cell cycle in primary neural precursor cells. previous studies were in tumor cells, which through the of the cell cycle in a the G1 phase of the cell cycle may be in this cell type. In primary cells, it that both CDK4/6 and CDK2 activity is to the cells to undergo S The of independent would in primary cells for the their We for and Lee for of this

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