From Cell Cycle to Differentiation

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Feb 1, 2006 - In human leukemia cell lines, p21 inhibited cdk4 and cdk2 activity but it did not ..... Durand B, Fero ML, Roberts JM, Raff MC. p27Kip1 alters the ...
[Cell Cycle 5:3, 266-270, 1 February 2006]; ©2006 Landes Bioscience

From Cell Cycle to Differentiation Perspective

An Expanding Role for Cdk6

ABSTRACT

*Correspondence to: Philip W. Hinds; Molecular Oncology and Research Institute; Department of Radiation Oncology; Tufts-New England Medical Center; 750 Washington Street #5609; Boston, Massachusetts 02111 USA; Email: phinds@ tufts-nemc.org Received 12/02/05; Accepted 12/06/05

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2385

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Oncology and Research Institute; Department of Radiation Oncology; Tufts-New England Medical Center; Boston, Massachusetts USA

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1Department of Biology; Connecticut College; New London, Connecticut USA

Over ten years ago, cdk6 was identified as a new member in a family of vertebrate cdc-2 related kinases.1 This novel kinase was found to partner with the D-type cyclins and to possess pRb kinase activity in vitro.1 Recently, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. Since exit from the cell cycle is a necessary step in the process of differentiation, it may not seem surprising that downregulation of a mitogenic factor may be required for this process. It is, however, surprising that this association has not been previously uncovered and that it is apparently not shared with cdk4, long understood to be a functional homolog of cdk6. As this story unfolds it will be important to discover if the role of cdk6 in differentiation is pRb-dependent or pRb-independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation.2

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Martha J. Grossel1 Philip W. Hinds2,*

The G1-phase kinases, cdk4 and cdk6, share 71% amino acid identity, and both partner with all three D-type cyclins to phosphorylate pRb in vitro.1,3-6 In their work identifying cdk6, Meyerson and Harlow suggested that further experiments should determine whether cdk6, or cdk4, is the physiological pRb kinase.6 Evidence gathered over the years has suggested that these kinases function redundantly in G1 phase of the cell cycle as proteins that, when complexed with D-type cyclins, phosphorylate pRb (reviewed in ref. 7). Recent studies indicate that the three D-type cyclins (D1, D2, D3) might differently impact some pRb functions and that the cyclin D family may not be completely functionally redundant.8,9 Because the D cyclins and their associated kinases are responsive to mitogenic signals, they are uniquely positioned to regulate cell cycle progression. Cdk4 and cdk6 both phosphorylate pRb but poorly phosphorylate Histone H1, the preferred in vitro substrate of cdk2,4,5,10,11 and both are expressed ubiquitously, (ref. 6 and Sicinski P, personal communication). Initial experiments that identified high levels of cdk6 activity only in T cells suggested that cell-type specific expression might explain the need for two distinct pRb family kinases. However, the more recent understanding that both kinases are expressed in most cell types, suggests that these kinases have discrete functions. Recent studies of cdk6-knockout mice indicate that mice lacking cdk6 are viable and develop relatively normally, however these mice did contain defects in hematopoesis including decreased cellularity of the thymus, of red blood cells and of lymphocytes.12 In addition, females were reduced in size and one-third of females were sterile.12 The mild phenotype of the cdk6 knockout suggests that cdk6 functions may be compensated for either by cdk4, or possibly by cdk2, which has been observed in association with D-type cyclins in cdk4/cdk6 double knockout mice.12 While a double knockout of cdk2 and cdk6 did not exacerbate developmental defects, mice lacking both cdk4 and cdk6 die in embryonic development due to severe anemia, supporting a hypothesis of functional compensation between cdk4 and cdk6.12 Functional compensation is not a new paradigm for cell cycle regulators-for example, the pocket proteins p130, p107 and pRb are commonly believed to have distinct functions, but in mouse cells with compromised pRB function, p130 and p107 are important cell cycle regulatory factors.13

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Martha J. Grossel is supported by NSF Grant number 9984453 Philip W. Hinds is supported by CA096527 and DE015302.

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ACKNOWLEDGEMENTS

CHARACTERIZATION OF cdk6

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cdk6, differentiation, G1 phase, pRb, cdk4, cyclin D

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KEY WORDS

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DISTINCT FUNCTIONS OF Cdk4 AND Cdk6 For a decade or more, differences in cdk4 and cdk6 have been identified in a variety of experimental models. For instance, Lucas and coworkers found that in T cells, activation of cdk6 preceded cdk4 activation by several hours.14,15 More recently, distinct responses of cdk4 and cdk6 to inhibitors of the cell cycle have been observed. In human leukemia cell lines, p21 inhibited cdk4 and cdk2 activity but it did not inhibit the pRb kinase activity of cdk6, despite demonstrated binding of p21 to the kinase.16 Recent studies have also shown that cdk4 and cdk6 phosphorylate pRb with different residue selectivity in vitro. Cdk4 preferentially phosphorylates the threonine residue at amino acid 826 on pRb while cdk6 preferentially phosphorylates threonine 821, suggesting that the cdk itself may contribute to substrate recognition.17 Finally, Fbxo7, an F box protein, was shown to specifically bind cdk6 over cdk4 and Fbxo7dependent transformation of murine fibroblasts required cdk6.18 Studies of diverse tumor types also suggest functional differences in these kinases: Cdk4 is specifically targeted in human melanoma,19,20 while cdk6 activity has been found to be elevated in squamous cell carcinomas21 and neuroblastomas,22 without alteration of cdk4 activity. In addition to differences in expression patterns in tumors, cdk4 and cdk6 also demonstrate distinct patterns of subcellular localization. Cdk6 was localized predominatly in the cytoplasm of U2OS cells23 and also in mouse astroctyes where cdk4 was localized to the nucleus.24 In T cells, cdk6 was detected in both the nucleus and cytoplasm, but only the nuclear fraction was active as a pRb kinase.25 Cdk6 has even been localized to the ruffling edge of spreading fibroblasts, suggesting a function for cdk6 in controlling cell spreading.26 Differences in subcellular localization could indicate that unique substrates of cdk4 and cdk6 may be found in some cell types. Recently, Smad3 was found to be a substrate of both cdk4 and cdk2 and phosphorylation of Smad3 by these kinases inhibited its transcriptional activity.27 While cdk6 was not studied in this published work, Smad3 may prove to be the first of several novel substrates of the G1 kinases.

Cdk6 BLOCKS DIFFERENTIATION

Recent findings demonstrate a new role for cdk6 in the differentiation of a variety of cell types. This function is not shared with cdk4. One of the earliest reports suggesting a role for cdk6 in differentiation was reported in 2000.28 In this work, the entry of murine erythroid leukemia (MEL) cells into terminal differentiation was accompanied by a decline in the activity of cdk2 followed by a decline in cdk6 activity. Inhibitors that blocked the combination of cdk2 and cdk6 triggered differentiation while inhibition of the combination of cdk2 and cdk4 did not. This work was later expanded to clearly show that MEL cells expressing an inhibitor-resistant form of cdk6 (cdk6R31C), but not an inhibitor-resistant form of cdk4 (cdk4R24C) blocked DMSO-induced differentiation of MEL cells.29 It was shown concomitantly that expression of cdk6 in mouse astrocytes was associated with dedifferentiation of astrocytes to glial progenitor-like cells.24 Expression of cdk6, but not cdk4 expression, in mouse astrocytes resulted in a drastic morphology change and in changes in patterns of expression of well-characterized markers of glial differentiation.24 More recently, studies indicated that BMP-2-stimulated osteoblast differentiation was inhibited by overexpression of cdk6 but not cdk430 and that cdk6 protein levels, www.landesbioscience.com

but not those of cdk4, were downregulated by RANKL-induced osteoblast differentiation of murine monocytic RAW cells.31 In osteoblast differentiation, the inhibition of differentiation by cdk6 expression was declared independent of its role in cell cycle regulation since cell cycle changes were not observed.30 The mechanism by which cdk6 expression exerts its effect on differentiation is now beginning to be uncovered. Surprisingly, studies have suggested a role for cdk6 in halting cell proliferation: While this result would be expected to precede differentiation, it is a novel role for a protein that has long been considered mitogenic. In one study, NIH3T3 cells overexpressing cdk6 showed a reduced growth rate compared to parental 3T3 cells, possibly through a mechanism involving the accumulation of p53 and p130 growthsuppressing proteins.32 A similar effect was seen in breast tumor cell lines. Cell lines transfected with cdk6 showed a reduced rate of proliferation compared with parental tumor cell lines and, interestingly, normal mammary epithelial cells had a high level of cdk6 protein while breast tumor-derived cell lines had much lower levels of cdk6.33 Because cells in higher organisms only cease dividing at the end of the process of differentiation, tight regulation of cell proliferation is an important part of the process that results in terminal differentiation.34 In a search for the mechanism of cdk6 function in differentiation, factors upstream and downstream are beginning to be uncovered. For instance, the transcription factor PU.1, which is downregulated in MEL differentiation35 may regulate the synthesis of cdk6 mRNA,29 as might Bone Morphogenic Protein 2 (BMP-2). BMP-2 is a signaling molecule that stimulates osteoblast differentiation through Smad mediator proteins. In BMP-2-induced osteoblast differentiation, cdk6 was downregulated by BMP-2/Smad mediated transcriptional repression and this downregulation of cdk6 was required for differentiation.30 In a related study of bone differentiation, cdk6 overexpression prevented the transcription factor, Runx2, from loading on the osteocalcin promoter, but did not exclude pRb, a Runx2 cofactor, from the promoter.31 These experiments may point toward a mechanism of cdk6 function. In the bone system, cdk6 may influence factors that prevent Runx2-mediated transcription on the osteocalcin promoter. This function appears to be independent of pRb, which appears to stay on the promoter even when its cofactor Runx2 does not load.30 Other systems also point to transcriptional mechanisms of cdk6 function in differentiation. In the astrocyte model, elevated levels of Id4 protein coincided with a change in differentiation status of cultured mouse astroyctes that expressed cdk6.24 Id4 is a member of the Id family of proteins (for Inhibitor of Differentiation). These proteins inhibit differentiation by forming heterodimers with transcriptional regulatory proteins (reviewed in ref. 36). Overexpression of Id genes can inhibit the differentiation of B lymphocytes, muscle cells, mammary epithelial cells, erythroid cells and oligodendrocyte precursor cells.37 Published studies in several systems now suggest that transcriptional regulation may be involved in the effect of cdk6 on the process of differentiation. In the past two years, excess expression of cdk6 has been shown to inhibit differentiation of mouse erythroid leukemia cells and has been found to be associated with a less-differentiated state in mouse astrocytes. In addition, osteoblast differentiation required downregulation of cdk6 and RANK-L mediated differentiation of osteoclasts resulted in reduced levels of cdk6 protein. Thus, cdk6 expression patterns have been shown to affect differentiation and, conversely, differentiation has been shown to influence cdk6 levels in multiple cell types and in a wide variety of experimental designs.

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Figure 1. A model for cdk6-regulated control of differentiation. Based on a model for phosphate regulation in yeast.60 Cdk6 regulates activation of genes involved in differentiation. In conditions where INK proteins exceed cdk6 concentration, Rb and its tissue-specific coactivators turn on transcription of differentiation genes. Under conditions where cdk6 exceed levels of inhibitor, cdk6 functions to phosphorylate and thereby inactivate transcription factors that drive expression of differentiation genes.

CELL CYCLE INHIBITORS AND DIFFERENTIATION

A PRB-DEPENDENT OR INDEPENDENT EVENT?

Although specific roles for cdk6 in differentiation are only now becoming clear, the CIP/KIP family of proteins including p21, p27 and p57 have long been studied as regulators of differentiation. Because the CIP/KIP proteins are important regulators of cdk6 activity, it is interesting to consider the contribution of these inhibitors to the process of differentiation. In 1995, several papers were published simultaneously that suggested that the cell cycle inhibitor protein, p21, halted cell proliferation during differentiation of muscle cells (see ref. 38). The idea that p21 might couple differentiation and cell cycle inhibition was attractive, but knockout studies in mice have complicated this simple model: mice lacking p21 undergo normal development and have no gross alterations in their organs.39,40 Mice nullizygous for p27 also did not demonstrate dramatic defects in tissue differentiation.41-43 Interestingly, p27 deficient mice demonstrated increased levels of p21 protein, raising the possibility that in mouse knockouts, remaining proteins can compensate for the deleted inhibitor. Despite the findings in knockout mice, the p21 and p27 proteins have proven to be important components of the differentiation process in some cell types. Oligodendrocyte progenitor cells from p27-/- mice differentiate poorly44 and studies in oligodendrocyte precursor cells indicate that p27 may be a part of an intrinsic timer of cell division that arrests the cell cycle and induces differentiation at the appropriate time.45 The p27 protein has been shown to initiate the differentiation of glial cells in the Xenopus retina46 and increased levels of p27 induced Central Glia-4 cells to differentiate into astrocytes.47 The third member of the CIP/KIP family, p57, has also been implicated in changes in proliferation and differentiation.48 Thus, all three CIP/KIP family members function in differentiation. The mechanism by which the CIP/KIP proteins function in differentiation is beginning to be understood and the mechanism is likely more complex than simply cell cycle withdrawal via kinase inhibition. For instance, the p57 protein was shown to modulate the subcellular localization of LIMK, a serine/threonine kinase that is involved in the regulation of actin filaments.49,50 Also, p27 expression induced the migration of cultured hepatocytes while p27-deficient MEFs failed to migrate in cell culture migration assays.51 The role of p27 and p57 in these functions appears to be distinct from the role of these proteins as inhibitors of cell division. For instance, regions of p57 not involved in cdk-binding (termed QT domains) may be involved in LIMK localization,48 and the ability to mediate cell cycle arrest is not a requirement for p27 function in cell migration.51 Thus, the CIP/KIP proteins, long understood to be potent inhibitors of cell division, are now understood to regulate aspects of differentiation as diverse as actin dynamics and cell migration.

It has been proposed that pRb maintains the post-mitotic state, protects against apoptosis and induces late markers of differentiation.52 The retinoblastoma protein has been shown to promote terminal differentiation of several cell types including myocytes, adipocytes, neurons and chondroctyes.2,53,54 Indeed, pRb knockout mice exhibit pronounced defects in erythroid, neuronal and lens development. In mice lacking pRB, these lineages were able to initiate differentiation but did not fully differentiate, suggesting that pRb might maintain cell cycle withdrawal that precedes expression of tissue-specific genes.55 However, recent data suggest that pRb may instead have a role in cell proliferation that follows differentiation. The idea that proliferative cells cannot stably differentiate has been supported by studies of mice lacking p27 or p19INK4d. These mice showed increased hair cell proliferation as might be expected in the absence of cell cycle inhibitors, but many of these cells underwent apoptosis stochastically well after differentiation, suggesting that terminal differentiation and proliferation may be incompatible in this compartment. However, additional studies suggest that an irreversible exit from proliferation is not required for “terminal” differentiation. In studies of embryonic mouse tissue lacking pRB in hair cells of the inner ear, differentiated, functional hair cells continued to cycle and divide.56 Thus, in a refinement of our understanding, pRB may not have a large effect on differentiation, but rather may act predominantly to enforce cell cycle exit coordinate with differentiation. Thus, different tissues may have different uses for pRB in terminal differentiation, but in all cases, the capacity to proliferate whether differentiated or not is increased through loss of functional pRB. The retinoblastoma protein is also directly involved in the expression of tissue-specific genes required for terminal differentiation. For example, pRb may activate the MyoD family of transcription factors2 and pRb may act as a transcriptional coactivator with the osteoblast transcription factor, Cbfa1/Runx2, in osteogenic differentiation.57 Differentiation-specific transcription factors may also act in opposition to the function of pocket proteins. For example, Id-2 can bind and inactivate pRb and loss of Id-2 was able to rescue differentiation defects in the nervous systems of Rb-null embryos.58 The transcription factor PU.1 has also been shown to interact with pRb through binding of its activation domain.59 Increasingly, data support the interplay of pRb and possibly other cell cycle regulators and the tissue-specific transcription factors that control differentiation. It will be of great interest to understand if the role of cdk6 in differentiation is pRb-dependent or pRb-independent. One recent clue suggests that pRb is unlikely to be the only mediator of cdk6driven inhibition of differentiation as no correlation was seen between the binding of pRb to the osteocalcin promoter and the cdk6induced block to differentiation in these cells.30 However, these data still allow the possibility that cdk6 may regulate the association of

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transcriptional coactivators that function with pRb on the promoter (see Fig. 1). The retinoblastoma protein and cdk6 may together provide a link between the complex cellular processes of differentiation and proliferation and in this way maintain the delicate balance between cellular division and differentiation.

CONCLUSIONS

Recent evidence seems to indicate that rather than being linked, differentiation and cell cycle regulation may in fact be independently regulated, simultaneous events. These events may involve dual roles for cdk6, pRb and cell cycle inhibitors, in both the cell cycle withdrawal and transcriptional regulation necessary for differentiation. Recent data suggests that pRb may play a direct role in activating differentiation-specific transcription and, as shown in Figure 1, this transcriptional role of pRb could be regulated by cdk6. For instance, pRb may provide a docking site for transcriptional activating proteins that are regulated by cdk6 phosphorylation. The role of cdk6 in differentiation may be pRb independent, or perhaps the distinction between cdk6 and cdk4 in this regard is the differential phosphorylation of pRb by these two kinases. Knockout studies of cdk6 have been difficult to interpret because these studies alter the stoichiometric relationship between kinases, cyclins and inhibitors. Perhaps a study of kinase-inactive and inhibitor-resistant cdk6 mutants will shed light on the mechanism of action of cdk6 in the complex process of differentiation. It will be interesting to learn if cdk6 mimics known mechanisms of differentiation such as activating transcription factors or affecting actin dynamics, or if it will reveal an entirely new function to regulate the process of differentiation. References 1. Meyerson M, Enders GH, Wu C, Su L, Gorka C, Nelson C, Harlow E, Tsai L. A family of human cdc2-related protein kinases. EMBO 1992; 11:2909-17. 2. Yee AS, Shih HH, Tevosian SG. New perspectives on retinoblastoma family functions in differentiation. Front Biosci 1998; 3:D532-47. 3. Kwon TK, Buchholz MA, Gabrielson EW, Nordin AA. A novel cytoplasmic substrate for cdk4 and cdk6 in normal and malignant epithelial derived cells. Oncogene 1995; 11:2077-83. 4. Matsushime H, Ewen M, Strom D, Kato J, Hanks S, Foussel M, Sherr C. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D Type G1 cyclins. Cell 1992; 71:323-34. 5. Matsushime H, Quelle DE, Shurtleff SA, Shibuya M, Sherr CJ, Kato J. D-type cyclindependent kinase activity in mammalian cells. Mol Cell Biol 1994; 14:2066-76. 6. Meyerson M, Harlow E. Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Mol Cell Biol 1994; 14:2077-86. 7. Ekholm SV, Reed SI. Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle. Curr Opin Cell Biol 2000; 12:676-84. 8. Baker GL, Landis MW, Hinds PW. Multiple functions of D-type cyclins can antagonize pRb-mediated suppression of proliferation. Cell Cycle 2005; 4:330-8. 9. Paternot S, Arsenijevic T, Coulonval K, Bockstaele L, Dumont JE, Roger PP. Distinct specificities of pRb phosphorylation by CDK4 activated by cyclin D1 or cyclin D3: Differential involvement in the distinct mitogenic modes of thyroid epithelial cells. Cell Cycle 2006; In press. 10. Ewen ME, Sluss HK, Whitehouse LL, Livingston DM. TGF beta inhibition of Cdk4 synthesis is linked to cell cycle arrest. Cell 1993; 74:1009-20. 11. Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev 1993; 7:331-42. 12. Malumbres M, Sotillo R, Santamaria D, Galan J, Cerezo A, Ortega S, Dubus P, Barbacid M. Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell 2004; 118:493-504. 13. Sage J, Mulligan GJ, Attardi LD, Miller A, Chen S, Williams B, Theodorou E, Jacks T. Targeted disruption of the three Rb-related genes leads to loss of G1 control and immortalization. Genes Dev 2000; 14:3037-50. 14. Lucas JJ, Szepesi A, Domenico J, Tordai A, Terada N, Gelfand EW. Differential regulation of the synthesis and activity of the major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4, during cell cycle entry and progression in normal human T lymphocytes. J Cell Physiol 1995; 165:406-16. 15. Lucas JJ, Szepesi A, Modiano JF, Domenico J, Gelfand EW. Regulation of synthesis and activity of the PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), a major cyclin D-associated cdk4 homologue in normal human T lymphocytes. J Immunol 1995; 154:6275-84.

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Zuo L, Weger J, Yang Q, Goldstein A, Tucker M, Walker G, Hayward N, Dracopoli N. Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma. Nature genet 1996; 12:97-9. 21. Timmermann S, Hinds PW, Munger K. Elevated activity of cyclin-dependent kinase 6 in human squamous cell carcinoma lines. Cell Growth and Differentiation 1997; 8:361-370. 22. Easton J, Wei T, Lahti J, Kidd V. Disruption of the cyclin D/cyclin-dependent Kinase/INK4/retinoblastoma protein regulatory pathway in human neuroblastoma. Cancer Research 1998; 58:2624-32. 23. Grossel MJ, Baker GL, Hinds PW. cdk6 can shorten G1 phase dependent upon the N-terminal INK4 interaction domain. J Biol Chem 1999; 274:29960-7. 24. Ericson KK, Krull D, Slomiany P, Grossel MJ. Expression of cyclin-dependent kinase 6, but not cyclin-dependent kinase 4, alters morphology of cultured mouse astrocytes. Mol Cancer Res 2003; 1:654-64. 25. Mahony D, Parry K, Lees E. Active cdk6 complexes are predominatly nuclear and represent only a minority of the cdk6 in T cells. Oncogene 1998; 16:603-11. 26. Fahraeus R, Lane DP. The p16(INK4a) tumor suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alphavbeta3 to focal contacts. EMBO J 1999; 18:2106-18. 27. Matsuura I, Denissova NG, Wang G, He D, Long J, Liu F. Cyclin-dependent kinases regulate the antiproliferative function of Smads. Nature 2004; 430:226-31. 28. Matushansky I, Radparvar F, Skoultchi AI. Reprogramming leukemic cells to terminal differentiation by inhibiting specific cyclin-dependent kinases in G1. Proc Natl Acad Sci USA 2000; 97:14317-22. 29. Matushansky I, Radparvar F, Skoultchi AI. CDK6 blocks differentiation: Coupling cell proliferation to the block to differentiation in leukemic cells. Oncogene 2003; 22:4143-9. 30. Ogasawara T, Kawaguchi H, Jinno S, Hoshi K, Itaka K, Takato T, Nakamura K, Okayama H. Bone morphogenetic protein 2-induced osteoblast differentiation requires Smad-mediated downregulation of Cdk6. Mol Cell Biol 2004; 24:6560-8. 31. Ogasawara T, Katagiri M, Yamamoto A, Hoshi K, Takato T, Nakamura K, Tanaka S, Okayama H, Kawaguchi H. Osteoclast differentiation by RANKL requires NF-kappaBmediated downregulation of cyclin-dependent kinase 6 (Cdk6). J Bone Miner Res 2004; 19:1128-36. 32. Nagasawa M, Gelfand EW, Lucas JJ. Accumulation of high levels of the p53 and p130 growth-suppressing proteins in cell lines stably overexpressing cyclin-dependent kinase 6 (cdk6). Oncogene 2001; 20:2889-99. 33. Lucas JJ, Domenico J, Gelfand EW. Cyclin-dependent kinase 6 inhibits proliferation of human mammary epithelial cells. Mol Cancer Res 2004; 2:105-14. 34. Coffman JA. Runx transcription factors and the developmental balance between cell proliferation and differentiation. Cell Biol Int 2003; 27:315-24. 35. Rekhtman N, Radparvar F, Evans T, Skoultchi AI. Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: Functional antagonism in erythroid cells. Genes Dev 1999; 13:1398-411. 36. Norton JD, Deed RW, Craggs G, Sablitzky F. Id helix-loop-helix proteins in cell growth and differentiation. Trends Cell Biol 1998; 8:58-65. 37. Kondo T, Raff M. The Id4 HLH protein and the timing of oligodendrocyte differentiation. EMBO J 2000; 19:1998-2007. 38. Marx J. Cell biology: Cell cycle inhibitors may help brake growth as cells develop. Science 1995; 267:963-4. 39. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 1995; 82:675-84. 40. Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 1995; 377:552-7. 41. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY, Nakayama K. Mice lacking p27 (Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 1996; 85:707-20. 42. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27 (Kip1). Cell 1996; 85:721-32. 43. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27 (Kip1)-deficient mice. Cell 1996; 85:733-44. 44. Casaccia-Bonnefil P, Tikoo R, Kiyokawa H, Friedrich Jr V, Chao MV, Koff A. Oligodendrocyte precursor differentiation is perturbed in the absence of the cyclin-dependent kinase inhibitor p27Kip1. Genes Dev 1997; 11:2335-46.

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45. Durand B, Fero ML, Roberts JM, Raff MC. p27Kip1 alters the response of cells to mitogen and is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation. Curr Biol 1998; 8:431-40. 46. Ohnuma S, Philpott A, Wang K, Holt CE, Harris WA. p27Xic1, a Cdk inhibitor, promotes the determination of glial cells in Xenopus retina. Cell 1999; 99:499-510. 47. Tikoo R, Casaccia-Bonnefil P, Chao MV, Koff A. Changes in cyclin-dependent kinase 2 and p27kip1 accompany glial cell differentiation of central glia-4 cells. J Biol Chem 1997; 272:442-7. 48. Zhang P, Liegeois NJ, Wong C, Finegold M, Hou H, Thompson JC, Silverman A, Harper JW, DePinho RA, Elledge SJ. Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome. Nature 1997; 387:151-8. 49. Yokoo T, Toyoshima H, Miura M, Wang Y, Iida KT, Suzuki H, Sone H, Shimano H, Gotoda T, Nishimori S, Tanaka K, Yamada N. p57Kip2 regulates actin dynamics by binding and translocating LIM-kinase 1 to the nucleus. J Biol Chem 2003; 278:52919-23. 50. Tanaka H, Homma K, Iwane AH, Katayama E, Ikebe R, Saito J, Yanagida T, Ikebe M. The motor domain determines the large step of myosin-V. Nature 2002; 415:192-5. 51. McAllister SS, Becker-Hapak M, Pintucci G, Pagano M, Dowdy SF. Novel p27(kip1) C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions. Mol Cell Biol 2003; 23:216-28. 52. Lipinski MM, Macleod KF, Williams BO, Mullaney TL, Crowley D, Jacks T. Cell-autonomous and noncell-autonomous functions of the Rb tumor suppressor in developing central nervous system. EMBO J 2001; 20:3402-13. 53. Cobrinik D, Lee MH, Hannon G, Mulligan G, Bronson RT, Dyson N, Harlow E, Beach D, Weinberg RA, Jacks T. Shared role of the pRB-related p130 and p107 proteins in limb development. Genes Dev 1996; 10:1633-44. 54. Hansen JB, Riele H, Kristiansen K. Novel function of the retinoblastoma protein in fat: Regulation of white versus brown adipocyte differentiation. Cell Cycle 2004; 3:774-8. 55. Lipinski MM, Jacks T. The retinoblastoma gene family in differentiation and development. Oncogene 1999; 18:7873-82. 56. Sage C, Huang M, Karimi K, Gutierrez G, Vollrath MA, Zhang DS, Garcia-Anoveros J, Hinds PW, Corwin JT, Corey DP, Chen ZY. Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 2005; 307:1114-8. 57. Thomas DM, Carty SA, Piscopo DM, Lee JS, Wang WF, Forrester WC, Hinds PW. The retinoblastoma protein acts as a transcriptional coactivator required for osteogenic differentiation. Mol Cell 2001; 8:303-16. 58. Iavarone A, Lasorella A. Id proteins in neural cancer. Cancer Lett 2004; 204:189-96. 59. Hagemeier C, Bannister AJ, Cook A, Kouzarides T. The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB. Proc Natl Acad Sci USA 1993; 90:1580-4. 60. Cross F. Transcriptional regulation by a cyclin-cdk. Trends Genet 1995; 11:209-11.

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