Rb inactivation in cell cycle and cancer - ULB

Perspective

Perspective

Cell Cycle 9:4, 689-699; February 15, 2010; © 2010 Landes Bioscience

Rb inactivation in cell cycle and cancer The puzzle of highly regulated activating phosphorylation of CDK4 versus constitutively active CDK-activating kinase Sabine Paternot, Laurence Bockstaele, Xavier Bisteau, Hugues Kooken, Katia Coulonval and Pierre P. Roger* Institute of Interdisciplinary Research (IRIBHM); Faculté de Médecine; Université Libre de Bruxelles; Brussels, Belgium

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yclin-dependent kinase (CDK) 4 is a master integrator that couples mitogenic/oncogenic signalling cascades with the inactivation of the central oncosuppressor Rb and the cell cycle. Its activation requires binding to a D-type cyclin and then T-loop phosphorylation at T172 by the only identified CDKactivating kinase in animal cells, cyclin H-CDK7. In contrast with the observed constitutive activity of cyclin H-CDK7, we have recently identified the T172phosphorylation of cyclin D-bound CDK4 as a crucial cell cycle regulatory target. Intriguingly, the homologous T177-phosphorylation of CDK6 is weak in several systems and does not present this regulation. In this Perspective, we review the recent advances and debates on the multistep mechanism leading to activation of D-type cyclin-CDK4 complexes. This involves a re-evaluation of the implication of Cip/Kip CDK “inhibitors” and CDK7 in this process. CDK4 and the Cell Cycles

Key words: CDK4, CDK6, p27, CAK, CDK7, cell cycle signalling Submitted: 10/23/09 Revised: 11/12/09 Accepted: 11/12/09 Previously published online: www.landesbioscience.com/journals/cc/ article/10611 *Correspondence to: Pierre P. Roger; Email: [email protected]

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In higher eukaryotes, the successive cell cycle phases are largely orchestrated by various cyclin-cyclin-dependent kinase (CDK) complexes. Nevertheless, genetic ablation in mice of different combinations of cell cycle cyclins and CDKs has shown that most of them are dispensable for mouse development, unveiling partly overlapping functions and unexpected compensatory mechanisms.1-3 The activity of CDK1 alone suffices to drive the mammalian cell cycles associated with early embryonic development in all cell lineages.4

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That does not mean that other CDKs and cyclins are destined to play only accessory roles, that cells will not become “addicted” to these regulators once they exert their important function.5 Indeed, differentiation and installation of non-proliferative states during development progressively impose additional cell-specific regulations, which involve replacement of most interphase functions of CDK1 by CDK2 or CDK4 and its close homolog CDK6. CDK2, which is activated first by cyclin E at G1/S transition and then cyclin A during S-phase progression, thus appears more professionally involved in both firing and termination of DNA replication.6,7 CDK4 and CDK6, activated by the three D-type cyclins, have a very restricted substrate selectivity and are specialized to phosphorylate and inactivate the central transcriptional cell cycle controller Rb and related p107 and p130 proteins.8,9 This function is indeed essential since the CDK4/6 inhibitor p16 or neutralization of cyclin D1 do not prevent cell cycle progression in Rb-defective cells.10-12 Recently, the concept has emerged that Rb phosphorylation by CDK4/6 leads not only to critical E2Fdependent transcription of essential cell cycle enzymes and regulators13 but also to assembly of the pre-replication complex in G1 phase.14,15 Genetic ablation of both CDK4 and CDK6,16 or of the three D-type cyclins17 permits cell cycles involved in development of most cell lineages. Nevertheless, in some adult differentiated tissues, lack of CDK4 or individual D-type cyclins has been found to severely impair cell cycle progression. Cyclin D1 is required for

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retinal development and pregnancy-induced mammary hyperplasia,18 and cyclin D2 is required for FSH-induced gonadal cell proliferation.19 Development of insulin-producing β-cells is much reduced in CDK4 knock-out mice.20 Other studies have shown that CDK4 is indispensable for postnatal proliferation of somato/lactotrophs21 and Schwann cells,22 whereas it is dispensable for prenatal development of these cells. Similarly, cyclins D2 and D1 are essential for postnatal but not prenatal pancreatic beta-cell growth.23 Another interesting example is the appearance of the cell cycle requirement for cyclin D3 associated with the maturation of T lymphocytes24 or late stages of neutrophil development.25 This indeed illustrates the installation of new cell cycle controls associated with new exigencies of tissue homeostasis during late preand post-natal development and adulthood. In most tested cells in culture that express normal Rb function, inhibition of CDK4/6 activity by the p16INK4A CDK4/6 inhibitor10,12,26 or neutralization of D-type cyclins27-30 potently inhibit proliferation. Ablation of both CDK4 and CDK6 or of the three D-type cyclins leads to late embryonic lethality,16,17 which precludes evaluation of their combined requirement for normal tissue homeostasis in adult animals. Therefore, the conclusion that in adult animals CDK4 and CDK6 are only required for the proliferation of some specialized cell types2 is not supported by experimental evidence. Inactivation of Rb by at least one of the six possible D-type cyclin-CDK4/6 complexes might still have at least a facilitative, if not essential, role in supporting proliferation of most normal differentiated cells. CDK4 activity is deregulated in many human tumors.31-33 It has been demonstrated to be absolutely crucial for various oncogenic transformation processes,2,17,34-39 suggesting that many cancer cells might be addicted to high CDK4 activity. Deregulation does not only result from oncogenic hyperactivation of mitogenic signalling cascades ending at D-type cyclin gene transactivation. Every particular component of “the Rb pathway” act as tumor suppressors or protooncogenes (reviewed in refs. 31, 32 and 40). Amplification or rearrangement

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of the genes encoding cyclins D1, D2 and D3 have been found in many human cancers and leukemias. Oncogenic mutations of cyclin D1 enforcing its nuclear accumulation have been described.33,41 Loss of p16 by mutation, deletion or gene silencing is extremely frequent. CDK4 itself is overexpressed as a result of gene amplification, or subject to a mutation that renders it insensitive to p16. Interesting models of CDK4/6 deregulation are also provided by some oncogenic viruses. Cyclin K encoded by Kaposi’s sarcoma-associated herpesvirus strongly activates CDK6 and CDK4 through original mechanisms,42 and the Tax oncoprotein of human T-cell leukaemia virus type 1 directly binds to and activate CDK4, which involves enhanced cyclin D interaction and suppression of inhibition by p16.43,44 By determining inactivation of the central oncosuppressor Rb, CDK4/6 activity might thus be one of the most common essential features for cancer cell proliferation, justifying its current interest as one of the most promising therapeutic target.45 CDK4 Regulation: A Complex Multistep Process Such a widespread deregulation of CDK4 in cancers underscores the necessity to fully understand the various mechanisms involved in its activation process. As initially considered, mitogens activate CDK4/6 by inducing at least one D-type cyclin to concentrations allowing to overcome an inhibitory threshold imposed by INK4 CDK4/6 inhibitory proteins.46 However, increased expression of a D-type cyclin is clearly not sufficient to activate CDK4. As we have thoroughly reviewed it,47 activation of CDK4 is a peculiarly complex process requiring different successive steps, which are distinctly regulated. Such a complexity allows CDK4 to function as a master integrator of various mitogenic and antimitogenic controls of the cell cycle. Both the assembly of D-type cyclin-CDK4 complexes,30,48-51 their subsequent nuclear translocation30,52-57 and their catalytic activity can be subject to independent regulations. This has been well exemplified by the exploration of CDK4 activation in thyroid epithelial cells in primary culture induced to proliferate

by their physiological hormone TSH acting via cAMP.30,52,59-63 Both the assembly, nuclear import and modulation of activity of D-type cyclin-CDK4 complexes have been intimately related to their association with the Cip/Kip CDK “inhibitors” p21 and p27, which, at variance with initial studies,64 are now accepted to have both positive and negative influences on CDK4 complexes.47,65 Whereas it is very clear that binding to either p27 or p21 do stabilize D-type cyclin-CDK4 complexes in the nuclear compartment,52,54,63,66-68 their impact on the assembly and activity of these complexes remains a topic of endless debates and controversies.47,64,65,69-71 In this Perspective, we will not re-discuss the claimed assembly activity of p27 and p21,50,67,72 (in our experiments, cyclin D3-CDK4 assembly does not require Cip/ Kip proteins,52,68 in agreement with observations from p27/p21 knockout cells73,74). This subject has been extensively covered by our previous review.47 Here we focus on mechanisms specifically regulating the kinase activity of assembled D-type cyclin-CDK4, the recent evidence that it depends on regulated phosphorylation of CDK4 in its activation loop, and we discuss the implication of p27 and the CDK-activating kinase (CAK) in this process. Unexpectedly, regulation at this level selectively concerns CDK4 but not CDK6. The activity of D-type cyclin-CDK4 complexes is regulated independently of their assembly. The assembly of D-type cyclin-CDK4/6 complexes and even their nuclear location are not sufficient for their catalytic activity. Upon mitogenic stimulation of quiescent cells, appearance of the Rb-kinase activity of CDK4 is often delayed compared to the formation of D-type cyclin-CDK4 complexes.53,68,75-77 Both processes are clearly dissociated in other situations, which include the inhibition of the activity of D-type cyclinCDK4 complexes by cAMP in mouse macrophages78 and thyroid carcinoma cell lines,79 contact inhibition in 3Y1 rat fibroblasts80 and mink Mv1Lu cells,81 senescence of human fibroblasts,82 antiestrogen treatment of MCF-7 breast cancer cells,83 calmodulin-dependent kinase inhibitors in human fibroblasts,84 and arrest of cAMP-dependent mitogenic stimulation

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in dog thyrocytes by forskolin deprivation,60,62 TGFβ,61 inactivation of Rho proteins85 or mTOR inhibition by rapamycin (Blancquaert et al. submitted). Inhibition by p21 or p27 is the most generally proposed mechanism to explain the inactivity of D-type cyclin-CDK4 complexes.64,69,86 Namely, the activation of cyclin D1-CDK4 complexes during G1 phase progression depends on p27 disappearance,75,77 and is prevented by upregulation of p27,78,80 or p21.82,83 This obviously contrasts with the positive roles (stabilization and nuclear anchoring) of these “inhibitors” on D-type cyclin-CDK4 complexes, and the frequent observation that they can support the Rb-kinase activity of CDK4.65 Whereas G1 arrest by cAMP in mouse macrophages is associated with p27 upregulation which inhibits cyclin D1-CDK4 activity,78 in the cAMP-dependent G1-phase progression of dog thyrocytes, an apparently similar elevation of p27 concentration allows the nuclear import and activation of cyclin D3-CDK4 complexes, and both processes are prevented by TGFβ which inhibits the binding of this complex to p27.61 Similarly, inhibition of cyclin D1-CDK4 activity has been explained either by increased83 or decreased87 binding of p21. Such opposite effects of p21 or p27 on the activity of D-type cyclin-CDK4 might depend on their binding stoichiometry (two or more molecules of p21/ p27 could bind and inhibit D-type cyclinCDK4, whereas complexes with only one p21/p27 molecule are fully active).67,68,88,89 Alternatively, Y88-phosphorylation of p27 in proliferating cells (which can be mimicked by incubating bacterially-produced p27 with insect cell lysates) has been interestingly suggested to switch p27 from an inhibitor to a non-inhibitor CDK4 binding partner,50,81 reminiscent of a similar impact on CDK2 activity.71,90 We believe that both mechanisms are not necessarily exclusive since (1) highly phosphorylated p27 produced in insect or CHO cells also supports or inhibits CDK4 activity depending on its relative concentration; 68 (2) p27 is not detectably Y-phosphorylated in those transfected CHO cells (our unpublished results in collaboration with Hengst L) while permitting p27-associated Rb kinase activity if expressed at low relative


levels; 68 and (3) Y-phosphorylations could only affect the inhibitory effects observed with high p27 concentrations (p27 concentrations used by Blain and coworkers were 10- to 100-fold higher than those that suffice to bind and inhibit CDK281). Moreover, without overexpression of BCR-Abl or Src-family tyrosine kinases, Y-phosphorylated p27 forms are very low abundance species81,90,91 that do not bind preferentially to CDK4,81 which would clearly limit their impact and hardly explains the full activity of p27-bound CDK4 complexes reported in previous studies.89 The impact on CDK4 activity of the abundant S10-phosphorylation of p27 remains to be fully explored. In many studies, modulations of the activity of D-type cyclin-CDK4 complexes are not easily explained by modifications of their association with p21 or p27.60,61,68,79,84,85,92 For instance, in dog thyrocytes the withdrawal of the cAMP stimulation rapidly arrests the entry of cells in S-phase and Rb phosphorylation but does not reverse the formation of nuclear cyclin D3-CDK4-p27 complexes.60,93 Even in p21/p27 knockout fibroblasts, serumdependent activation of cyclin D1-CDK4 complex follows by several hours its assembly.74 Another central theme is the regulation of CDK activity by various inhibitory and activating phosphorylations. By analogy with the major regulation of CDK2 and CDK1 by inhibitory Y15-phosphorylation, a phosphorylation of CDK4 at Y17 has been found in UV irradiation-induced G1 arrest,94 during cell arrest in quiescence95 or in response to TGFβ.96 At variance with Y15-phosphorylation of CDK1, Y17phosphorylation of CDK4 could not be performed by Wee1 or Myt1 but by the same Src-family tyrosine kinases97-99 that also would ironically phosphorylate p27 to activate CDK4. However, it is generally undetectable in the absence of a tyrosine phosphatase inhibitor (vanadate),68,99-101 indicating that it is very unstable. This inhibitory modification thus only affects a thin fraction of CDK4 molecules in most situations. Therefore, it cannot be biologically significant, and the activity of CDK4, unlike CDK2 and CDK1, cannot be regulated by cdc25 phosphatases.47 By contrast, as shown by pioneering studies by

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Sherr’s lab, the activity of CDK4 absolutely depends on its T172-phosphorylation in the activation T-loop,68,100,102 analogous to the T160/T161 phosphorylation of CDK2 and CDK1. CDK6 activity similarly requires its phosphorylation at T177.103,104 In higher eukaryotes, T-loop phosphorylation of CDKs, including CDK4,102 is generally accepted to be performed by the only identified CDK-activating kinase (CAK), the nuclear cyclin H-CDK7-Mat1 complex,105-108 which, frustratingly, has been found to be constitutively expressed and activated102,109 (see also below). T-loop phosphorylation of CDKs is thus assumed to passively result from CDK binding to a cyclin and subsequent nuclear import. Critical CDK4 regulation by T172phosphorylation: A tale of cell cycle regulation by cyclic AMP. Act I. The second messenger cyclic AMP (cAMP) has major, but very divergent, negative or positive effects on cell cycle progression in different cell systems, apparently precluding any generalization of its mechanisms.110 The very first example of a cell cycle regulation by T172-phosphorylation of CDK4 was provided by the investigation by Sherr’s group of the G1-phase arrest induced by cAMP in murine macrophages.78 cAMP did not affect the expression of cyclin D1 and its association with CDK4, but it inhibited the Rb-kinase activity of cyclin D1-CDK4 complexes. As this group was just demonstrating that the activity of CDK4 produced in insect cells critically depends on its T172-phosphorylation,100 they analysed the phosphorylation of CDK4 in cAMP-arrested cells and observed that the inhibition of CDK4 activity indeed correlated with the disappearance of the CDK4 phosphopeptide that contains the T172-phosphorylation.78 These authors did not observe a reduction of CAK activity in cAMP-arrested cells, but they found that the just cloned CDK inhibitor p27 was upregulated by cAMP. Finally they showed that addition of bacterially-produced p27 to CDK4 complexes impeded their in vitro activation by CAK, leading them to logically conclude that upregulation of p27 was the mechanism of CDK4 phosphorylation inhibition and cell cycle arrest.78 As endogenous CDK4 is a low abundance protein, the two-dimensional analysis of its tryptic 32P-labeled

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phosphopeptides might have been a rather heroic approach. It has not been repeated in other studies to our knowledge. For about one decade, CDK4 T172-phosphorylation was no longer studied directly, probably due to lack of easy experiment tool, and it was only inferred from CDK4 activity. Act II. Thyroid epithelium is often considered the best example of a positive regulation of cell cycle by cAMP and PKA activation in response to stimulation by the physiological hormone TSH or pathological hyperactivation of TSH receptors in various thyroid diseases.111,112 In primary cultures of canine thyrocytes, cAMP triggers and sustains G1 phase progression and induces DNA replication. In this unique differentiation-associated mitogenic process, the required D-type cyclin, cyclin D3, was paradoxically down modulated,30,59 while the CDK “inhibitor” p27 was upregulated,52,58 as found during cAMP-dependent G1 arrest in other systems. However, cAMP activated CDK4 by promoting the assembly of the cyclin D3-CDK4 complex,30,59 its stabilization in nucleus through its binding to nuclear p27,52 and finally the activation of this complex through T172-phosphorylation of CDK4.47,62,68,113 To demonstrate this last step, phosphorylation of CDK4 was evidenced by its separation by two-dimensional gel electrophoresis (isoelectric focusing and SDS-PAGE), which allowed to precisely evaluate the proportion of phosphorylated and non-phosphorylated forms of CDK4 in its different complexes.61,68 Upregulated p27 indeed appeared to play a positive role in the cAMP-dependent activation of cyclin D3-CDK4 since p27bound CDK4 was both active and phosphorylated61 and since TGFβ selectively inhibited the cAMP-dependent mitogenesis at least in part by impeding the binding of cyclin D3-CDK4 to nuclear p27.52 TGFβ also inhibited the cAMP-dependent activity of cyclin D3-CDK4 complexes by decreasing the phosphorylation of CDK4, even in complexes residually associated with p27.61,68 The disappearance of cyclin D3-CDK4 activity upon arrest of cAMP stimulation also was explained by the disappearance of cyclin D3-bound T172-phosphorylated CDK4 but not by a reduced formation of cyclin D3-CDK4-p27 complexes.62 Therefore,

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Figure 1. For figure legend, see page 693.

the activating phosphorylation of CDK4 integrates the opposite cell cycle controls by cAMP and TGFβ after formation of cyclin D3-CDK4 complexes, and p27 does not oppose this process. Act III. Whereas cAMP is a positive modulator for the proliferation of many normal differentiated cells, it often inhibits the proliferation of their derivate cancer cells.110 This is also the case in thyroid carcinoma cell lines.79 In one such cell line, cAMP mostly acted by inhibiting

the oncogenically activated ERK cascade, which led to upregulation of p27 and disappearance of cyclin D1. Nevertheless in three other thyroid carcinoma cell lines, this was not observed and cAMP inhibited the activity of unaltered cyclin D1-CDK4 and/or cyclin D3-CDK4 complexes by decreasing the phosphorylation of CDK4.79 The activating phosphorylation of CDK4 thus presented as the common target of the opposite cell cycle regulations by cAMP. However, at variance


Figure 1. Highly regulated activating phosphorylation of CDK4 despite constitutively CAK activity in human T98G glioblastoma cells. In these cells arrested by serum-deprivation or combined inhibition of mTOR and MEK/ERK pathways, cyclin D3-bound CDK4 is not T172-phosphorylated (A), but remains activatable by CAK (cyclin H-CDK7) (B). This lack of T172-phosphorylation in G0/G1-arrested cells is puzzling because these cells express active cyclin H-CDK7 complexes able to activate cyclin D3 complexes from the same cells (C). (A) Quiescent serum-deprived T98G cells were stimulated (or not) for 10 h with 15% serum with (+) or without (-) a combination of mTOR-raptor inhibitor rapamycin (Rapa) and MEK inhibitor PD184352 (PD18). Cell lysates were immunoprecipitated with anti-cyclin D3 antibody (IP cyc D3), separated by 2D-gel electrophoresis and CDK4 was immunodetected. Arrows indicate the position of the T172-phosphorylated form induced by serum. Rapamycin and PD184352 cooperate to block this effect. (B) Inactive co-immunoprecipitated cyclin D3-CDK4/6 complexes (IP cyc D3) from T98G cells treated as in (A), i.e., either serum-deprived (lanes 1 & 2) or stimulated with serum in the presence of rapamycin and PD184352 (lanes 4 & 5), were incubated in the presence of ATP with (lanes 2 & 5) or without (lanes 1 & 4) recombinant purified cyclin H-CDK7-Mat1 (CAK). The resulting activation of the cyclin D3-CDK4/6 complexes was then assayed by their Rb-kinase activity (by a second incubation in the presence of a Rb fragment and ATP). The cyclin D3 complexes were separated by SDS-PAGE and immunoblotted for detection of CDK4 and in vitro phosphorylated Rb fragment using a phosphospecific Rb antibody (Rb-kinase). Controls include the incubation with CAK followed by Rb-kinase assay of mock immunoprecipitations (IP IgG) from the same T98G cell lysates (lanes 3 & 6). After in vitro activation by CAK, cyclin D3-CDK4/6 complexes from G0/G1-arrested cells were as active (lanes 2 & 6) as those isolated from serum-stimulated cells without CAK activation (lane 7). (C) The activity of co-immunoprecipitated cyclin H-CDK7 complexes (IP cyc H) was evaluated from T98G cells treated as in (A) with or without serum and the combination of rapamycin and PD184352. In this assay, these cyclin H-CDK7 complexes (lanes 1–3 & 6), or the recombinant cyclin H-CDK7-Mat 1 complex (CAK; lane 5), were mixed with cyclin D3-CDK4/6 complexes immunoprecipitated from serum-deprived T98G cells (IP cyc D3), which were inactive (lane 4), or a similar mock immunoprecipitation from the same lysate (IP IgG; lane 7). After incubation in the presence of ATP, the resulting activation of the cyclin D3-CDK4/6 complexes was then assayed by their Rb-kinase activity as in (B). The mixtures were then separated by SDS-PAGE and immunoblotted. We detected (i) the presence of cyclin H (cyc H) and CDK7 co-immunoprecipitated by the cyclin H antibody from T98G cells or recombinant CAK complex, (ii) the presence of the substrate, i.e., cyclin D3-CDK4 complexes from quiescent T98G cells (CDK4), and (iii) the in vitro activation of these cyclin D3 complexes reflected by the phosphorylation at S780 of the Rb fragment (Rb-kinase). The capacity of T98G cell cyclin H-CDK7 complexes to activate cyclin D3-CDK4/6 is essentially constitutive (lanes 1–3) (slight variations of the activity are due to weak variations in the amount of immunoprecipitated cyclin H), which cannot explain the all-or-none regulation of CDK4 phosphorylation seen in (A). Part of these data are extracted from reference 92.

with the scenario in murine macrophages (Act I), p27 was unlikely involved since: (1) p27 levels were not increased by cAMP in two B-Raf-mutated cell lines; (2) p27 did not inhibit CDK4 activity and CDK4 phosphorylation, as observed during cAMP-dependent mitogenesis of normal thyrocytes; 61,68 (3) inhibition of CDK4 activity by cAMP was also observed in the very active p21 complexes that do not contain p27; (4) p27 knockdown was insufficient to preclude cAMP-induced cell cycle arrest in the four thyroid carcinoma cell lines.79 Regulated T172-phosphorylation of CDK4 might be a general mechanism. Direct regulation of CDK4 activity through its activating phosphorylation is not restricted to the control of cell cycle by cAMP. We also observed it during serum stimulation of G1 phase progression in normal human fibroblasts68 and thyrocytes,63,113 as well as in several glioma cell lines including T98G cells.68,92 In these p16-defective T98G cells, cyclin D3-CDK4/6 complexes were abundantly formed in serum-deprived cells, but inactive due to poor phosphorylation of both CDK4 and CDK6. Moreover, serum induced both the T172-phosphorylation and Rb-kinase activity of CDK4 (Fig. 1A and B). Interestingly, p27 is highly expressed in this cell line, and thus p27bound CDK complexes were inactive


even after serum stimulation. However more than 50% of p27-bound CDK4 was phosphorylated in response to serum,68 indicating once again that p27-binding does not impair CDK4 phosphorylation, and furthermore that inhibition of CDK4 by p27 is independent of CDK4 phosphorylation. In T98G and other glioma cell lines, we recently showed that inhibition of either the MEK/ERK pathway using the selective MEK inhibitor PD184352, or of mTOR-raptor using rapamycin, moderately inhibited phosphorylation of CDK4 bound to cyclin D1 or cyclin D3 and p27. When combined, both inhibitors abolished CDK4 phosphorylation (Fig. 1A and B), Rb phosphorylation and proliferation, but did not display a similar synergy on levels of cyclin D1, cyclin D3 or p27, and formation of their complex with CDK4.92 This finding provides first evidence that MEK and mTOR-dependent cascades might signal cell cycle progression through T172-phosphorylation of CDK4, which appears as a crucial node integrating these pathways. Rapamycin also partly inhibits the cAMP/PKA-dependent phosphorylation of CDK4 in thyrocytes (Blancquaert et al. submitted). Although CDK4 phosphorylation has been investigated only in a limited series of cell cycle regulation models, until now we have found no exception to the rule: DNA synthesis = Rb

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phosphorylation = CDK4 activity = CDK4 T172-phosphorylation. Phosphorylation of CDK4 in its activation loop might thus be the last rate-limiting step of the complex CDK4 activation process (Fig. 2). The Puzzle “One might wonder why a system would be built with a need for an activating phosphorylation if the activating kinase is constitutively active” (Mark J. Solomon in ref. 105). CAK (cyclin H-CDK7), which is also critically involved in the transcription machinery, is considered the only kinase responsible for the activation loop phosphorylation of CDK1, CDK2, CDK4 and CDK6. In all the examples detailed above, the presence and activity of cyclin H-CDK7 complexes were not regulated68,78,79,92 as generally observed.102,109,114,115 CAK can unequivocally phosphorylate and activate cyclinCDK4/6 complexes,68,89,102-104 including those coimmunoprecipitated from quiescent T98G cells.68,79,92 So, why were cyclin D3-bound CDK4 and CDK6 not phosphorylated in serum-starved or PD184352/rapamycin-arrested T98G cells, if these complexes can be activated by mixing them with cyclin H-CDK7 complexes from the same cells92 (Fig. 1C)?

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Parenthetically, such a paradox does not disprove the hypothesis that CAK would be the sole CDK4-activating kinase. Several studies have also pointed out the T160-phosphorylation of CDK2 as a direct target of treatments that prevent S-phase entry.115-118 However a chemical genetics approach has recently demonstrated CDK7 to be the only kinase responsible for CDK2 T160-phosphorylation, at least in human HCT116 cells.108 p27 is back again. If (anti)mitogenic signalling cascades do not regulate the activity of CAK, they might nevertheless modulate its substrate specificity or its access to CDK4. The hypotheses linking T172-phosphorylation of CDK4 and p27 have been discussed above. Recently, a new interesting variation of this theme has been proposed by Blain and coworkers.119 This in vitro study suggests that p27 permits phosphorylation of CDK4 (and also CDK2 and CDK6) by CAK only if phosphorylated at Y89 (but not Y88) residue, and proposes that Y89-phosphorylation of p27 by a yet to be determined tyrosine kinase would explain how p27-bound cyclin D1-CDK4 complexes could be competent for activation by CAK in proliferating but not in contact-arrested Mv1Lu cells.119 This is indeed an appealing model, but several observations question its biological relevance, at least in cells that do not overexpress tyrosine kinases. It is unlikely that it can explain the regulations of CDK4 phosphorylation that we observe: (1) despite high relative expression of p27, non-phosphorylated CDK4 and CDK6 from inactive cyclin D3 complexes in serum-deprived T98G cells were competent for productive phosphorylation and activation by cyclin H-CDK7 from different sources68,79,92 (Fig. 1B and C); (2) if the model of Blain and coworkers is correct, the proportion of phosphorylated CDK4 in p27-cyclin D-CDK4 complexes must match the proportion of Y89-phosphorylated p27. As already mentioned, without tyrosine kinase overexpression Y-phosphorylated p27 is at best a very low abundance species,90,91 even in proliferating MV1Lu cells,81 and this would especially apply to phosphorylation of Y89, which (unlike Y88) is not the main phosphoacceptor residue of the tested tyrosine kinases.90 The low abundance of

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Y89-phosphorylated p27 forms sharply contrasts with the massive proportion (30–80%) of T172-phosphorylated CDK4 in p27-containing complexes that we observed in a variety of situations,61,68,92 including in transfected CHO cells with high inhibitory expression of p27,68 which was not detectably Y-phosphorylated (our unpublished data in collaboration with Hengst L). The study by Blain and coworkers provides a thorough confirmation that binding of unphosphorylated p27 to CDK4, CDK6 and CDK2 complexes prevents their activating phosphorylation by cyclin H-CDK7.119 So, how could T172phosphorylated CDK4 be so enriched in p27-bound complexes in all the investigated cell systems and situations? 61,68 Interestingly, the Blain’s study also shows that the impairment of CDK4 phosphorylation by p27 is specific of cyclin H-CDK7. Binding of non-Y89-phosphorylated p27 did not impair CDK4 phosphorylation by the yeast monomeric CAK Csk1,119 reminiscent of a similar observation in the case of CDK6.103 The Csk1-activated p27-bound CDK4 complexes nevertheless remain inactive,119 an “artificial” situation which in fact exactly recapitulates what we observed with high inhibitory concentrations of p27 in T98G cells and transfected/infected CHO and insect cells.68 Other pieces of the puzzle that do not fit the “one-CAK-fits-all-CDKs” model. Phosphorylation of cytoplasmic CDK4. At variance with yeast Cak1,120 cyclin H-CDK7-Mat1 is a purely nuclear complex.109 It was thus unexpected that deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4-p27 in the cytoplasm without reducing its T172-phosphorylation.68 Differential regulation of CDK4 bound to cyclin D1 or cyclin D3. In dog thyroid primary cultures, TGFβ inhibited the cAMP-dependent T172-phosphorylation and activity of cyclin D3-CDK4-p27 complexes, but it did not affect the activation of CDK4 by growth factors,52,61 which mostly involved cyclin D1 and p21.63 In human thyroid primary cultures, growth factors stimulated the phosphorylation and activity of CDK4 bound to cyclin D1, but not the activity of abundant cyclin D3-CDK4 complexes, which were

specifically activated by TSH.113 In three of the four studied thyroid carcinoma cell lines, cAMP selectively inhibited the T172-phosphorylation and activity of cyclin D1-CDK4 complexes with little or no impact on cyclin D3-CDK4.79 How could cyclin H-CDK7 discriminate between those different CDK4 complexes in cells? Differential regulation of CDK4 and CDK6. In T98G cells, serum induced the T172-phosphorylation of cyclin D3-bound CDK4, but the T177-phosphorylation of CDK6 bound to cyclin D3 or cyclin D1 remained weak and not induced.68,104 Similarly, in CEM T-lymphoblasts, the weakly expressed CDK4 bound to cyclin D3 was abundantly phosphorylated, whereas the T177-phosphorylation was undetectable in much more abundant cyclin D3-CDK6 complexes.104 Cotransfection of cyclin D3 or cyclin D1 with CDK4 or CDK6 in various serum-cultivated human or rodent cell lines induced the abundant T172-phosphorylation and Rb-kinase activity of cyclin-bound CDK4, but not the T177-phosphorylation and activity of cyclin-bound CDK6.104 These unexpected observations suggest that CDK4 might be the main functional and regulated D-type cyclin-dependent kinase in most cells and that CDK6 might play a minor role. This might explain why in CDK2 knockout mice, genetic inactivation of CDK6 brings almost no additional phenotype,16 whereas CDK4 knockout dramatically reduces Rb phosphorylation and arrests embryonic development at mid gestation121 (but see ref. 122 for partly divergent conclusions). CDK4 and CDK6 are close structural and functional homologs. Both are in vitro substrates of cyclin H-CDK7.102,103 More precisely, cyclin D3-bound CDK6 is an even better in vitro substrate of cyclin H-CDK7 than cyclin D3-CDK4, as only the former could be phosphorylated in the presence of low Mg-ATP concentrations that did not suffice for CDK4 phosphorylation,68,104 which likely explains the previous failure by others to activate CDK4 by CDK7 in conditions that allowed productive phosphorylation of CDK6 and CDK2 complexes.77,103 How could cyclin H-CDK7 discriminate between CDK4 and CDK6 complexes in cells but not in vitro?


Figure 2. Multistep activation of CDK4. CDK4 activation requires (i) increased expression of a D-type cyclin (cyc D) to levels that overcome an inhibitory threshold imposed by INK4 inhibitory proteins (p16, p15, p18, p19), (ii) CDK4 binding to this cyclin, (iii) cyclin D-CDK4 nuclear import mediated by binding to a Cip/Kip protein (p21 or p27) and (iv) CDK4 T172-phosphorylation within the resulting complex by a still hypothetical CDK4-activating proline-directed kinase (CDK4AK?), which determines the Rb-kinase activity. These successive steps can be dissociated and are distinctly regulated by mitogenic signaling and its oncogenic perversions. Some of these steps can be constitutive or permanently stimulated, especially in cancer cells. Cip/ Kip proteins have a complex dual role, which depends at least in part on their concentration relative to levels of CDK4 and D-type cyclins. Low levels of p21 transiently induced by growth factors in many cells, or p27 induced by TSH and cAMP in the specific case of dog thyrocytes, stabilize D-type cyclin-CDK4 in the nucleus and support its activation. By contrast, anti-mitogenic signaling induces or maintains much higher levels of Cip/Kip proteins, which leads to binding of additional Cip/Kip molecules to cyclin-CDK4 complexes, impairing their activity, but not necessarily CDK4 activating phosphorylation. Diamond arrowheads represent inductions; the other dashed arrows are activations (+) or inhibitions (-). Green/red colors indicate the stimulating/inhibitory potential.

Recognition of CDKs by CDK7 does not depend on the sequence surrounding their phosphoacceptor site. Cyclin H-CDK7 is suggested to utilize two distinct modes to recognize its substrates.107,123-125 Depending on phosphorylation of its own T-loop, CDK7-cyclin H recognizes RNA polymerase II125 and likely a number of nonCDK substrates including DNA-binding transcription factors, through sequences surrounding the phosphorylation site characterized by a proline at +1 position relative to the phosphoacceptor residue, similar to most residues phosphorylated by CDKs.124 By contrast, phosphorylation of CDKs by CDK7 would not require its own T-loop phosphorylation,125 and CDK recognition by CDK7 would not depend on a consensus sequence around the phosphoacceptor site (which differs in various CDKs and in general does not present a proline at +1 position) but on remote substrate motifs.107,123,124,126 Such remote motifs might be masked or distorted in p27-CDK complexes,126 perhaps explaining how could binding of p27 impair CDK4 phosphorylation by cyclin H-CDK7, while still


allowing phosphorylation by Csk1 which might recognize the T-loop directly.119 But in cells, CDK4 T172-phosphorylation absolutely depends on the proline 173 residue! The divergent activating phosphorylations of CDK4 and CDK6 observed in cells were totally unexpected since they present a high degree of homology, including at key residues predicted to allow interaction with CDK7.126 The sequences surrounding the phosphoacceptor sites of CDK4 and CDK6 are also almost perfectly conserved on a segment of 36 aminoacids. However, CDK4 is clearly distinct as its T172 site is followed by a proline, whereas a serine follows the phosphorylated T177 residue of CDK6. Among other CDKs phosphorylated by CDK7, only CDK11 also possesses a similar proline at +1 position, but this has been regarded as not influencing its mode of recognition by CDK7.124 As the unique P173 site of CDK4 is perfectly conserved across evolution since echinoderms, we decided to substitute it by a serine as in CDK6 sequence or a histidine as in CDK2 and CDK1. As expected, the P173S/H mutations did not adversely affect T172-

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phosphorylation and activation of cyclin D3-CDK4 by cyclin H-CDK7 in vitro,104 confirming the CDK-CDK7 recognition model. In cells, P173S/H mutations did not affect binding of CDK4 to D-type cyclins and its subcellular localization. However they completely abrogated the T172-phosphorylation and Rb-kinase activity of CDK4, reproducing the paradoxical case of CDK6.104 The converse S178P mutation of CDK6, thus mimicking in CDK6 the phosphorylation segment of CDK4, generated in cells a dramatic activation of CDK6 caused by its almost complete T177-phosphorylation, which was even independent of cyclin-binding at variance with the situation observed in CDK4.104 Further studies should evaluate the oncogenic potential of this first CDK6 activating mutation. Our observations thus indicate that cells possess a robust proline-directed kinase activity able to selectively activate CDK4 even when it is overexpressed, by recognizing its T-P motif. Can CDK7 still be considered as the sole CDK4-activating kinase? The

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simplest hypothesis fitting all these puzzling observations would be that at variance with CDK2 and CDK1,108 CDK4 would not be activated in cells by cyclin H-CDK7, but by one or several prolinedirected kinase(s), which, similar to the yeast Cak1 and Csk1,120,127 would have their expression and/or activity exquisitely regulated, would have an overall subcellular location, and would directly recognize the T-loop of CDK4 and its crucial proline, irrespective of CDK4 binding to Cip/Kip proteins (Fig. 2). The hypothesis of multiple CAKs in animal cells is not novel.106,127-129 A distinct CDK4-activating kinase(s) would help to resolve the complex issue of the dichotomy and divergent constrains of the dual roles of CDK7 in cell cycle and general transcription.107 In yeasts and plants, several CAKs with distinct substrate specificities coexist,127,130,131 and in S. Cerevisiae the CDK7 ortholog (Kin28) phosphorylates RNA polymerase II but does not act as a CAK, a function performed by Cak1. Why should animal cells with their multiple CDKs be different? On the other hand, no mammalian Cak1/Csk1 ortholog has been identified by genome sequencing, and, so far, cyclin H-CDK7 remains the only mammalian kinase found to phosphorylate and activate cyclin-CDK4 complexes in vitro. Nevertheless, compared to CDK2, CDK4 seems to be a poorer substrate of cyclin H-CDK7,103 perhaps because some key residues involved in CDK2-CDK7 interaction are not conserved in CDK4,126 and/or because binding of cyclin D1/D3 does not rearrange the activation segment of CDK4, at variance with the conformation of cyclin A-CDK2.132,133 This might explain why efficient phosphorylation of CDK4 would specifically require a kinase that directly recognizes its T-loop T-P motif. Candidates for the hypothetical proline-directed CDK4-activating kinases are numerous. For instance, they might include dozens of members of several subfamilies of poorly characterized protein kinases with partial homologies with both CDKs and MAP kinases. Alternatively, our experiments do not completely exclude the possibility that CDK7 could still be the catalytic subunit of this highly regulated proline-directed CDK4-activating kinase in intact cells. In

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its first mode of recognition of non-CDK substrates, CDK7 is also targeted to T/S-P motifs.124 Whereas cyclin H-CDK7 coimmunoprecipitated from various cell lysates does phosphorylate CDK4 independently of its +1 proline and is constitutively active (which suggests that covalent modifications of cyclin H, Mat1 and CDK7, including its T170-phosphorylation,125 are not involved in CDK4 regulation),68,79,92,104 the possibility remains that in cells, CDK7 might act differently, depending on an interaction with unknown accessory proteins, which might not be preserved in CDK7 coimmunoprecipitations. Such highly regulated accessory proteins might mediate the complex regulations of CDK4 phosphorylation, including its differential regulation in cyclin D1 or cyclin D3 complexes. It even could not be excluded that CDK7 would not utilize cyclin H to activate CDK4 in cells. This alternative hypothesis is compatible with the only direct evidence that CDK7 would be the sole kinase responsible for CDK4 activation, as obtained by Matsuoka and coworkers from Sherr’s lab.102 They found that two antisera produced against CDK7 can completely deplete lysates of NIH-3T3 cells of CAK activity (indirectly deduced from Rb-kinase activity of activated CDK complexes) for both cyclin A-CDK2 and cyclin D2-CDK4.102 These authors did not formally exclude that CDK4 CAK might contain other catalytic subunits that are antigenically related to CDK7.102 If these experiments can be extended to other cell types, they suggest that CDK7 would be the catalytic subunit of the CDK4activating complex, but they provide no information about its involved regulatory subunits, which might well be specific to the regulated CDK4 activation. Further studies should equally envisage these alternative hypotheses: unknown regulated CDK4-activating kinases, or unknown accessory proteins/regulatory subunits of CDK7 specifically involved in regulated activation of CDK4. Conclusions and Perspectives At variance with the situation in CDK1 and CDK2 cyclin complexes, the activating T-loop phosphorylation, instead of dephosphorylation of inhibitory sites by

cdc25 phosphatases, is the critical step determining the activity of CDK4 (Fig. 2).47 In all the cell cycle regulation models that we have recently investigated, Rb phosphorylation and DNA replication onset perfectly correlate with CDK4 T172-phosphorylation but not with the concentration of any CDK4/6 regulatory proteins (cyclin D1, cyclin D3, p27, p21) that are most generally considered as endpoints of mitogenic and antimitogenic signal transduction cascades and their deregulations in cancer. The complexity of the direct regulation of CDK4 phosphorylation thus recapitulates the complexity of G1-phase controls. By contrast, CDK6 activating phosphorylation is not subjected to this regulation and remains weak, whereas CAK activity seems unrestricted for T-loop phosphorylation of CDK2 and CDK1 complexes. CDK6 might also be a poor CDK7 substrate in cells. Because of its specifically regulated and productive phosphorylation, CDK4 might thus be the main functional D-type cyclin-dependent kinase in most cells. Divergent results in CDK2 knockout mice, showing that CDK6 cannot,121 or can,122 functionally replace CDK4 to achieve Rb phosphorylation and late embryonic development, are perhaps to be explained by different levels of CAK activity. As CDK4 T172-phosphorylation is emerging as a cell cycle regulator determinant, major efforts should be devoted to the understanding of mechanisms responsible for its regulation. Recent determinations of crystallographic structure of D-type cyclin-CDK4 complexes have indicated that their structural activation mechanisms cannot be directly extrapolated from those of cyclin A-CDK2 complexes. Namely, at variance with the cyclin A-CDK2 complex, cyclin binding may not be sufficient to drive the CDK4 active site toward an active conformation,132,133 and it also does not preclude the accessibility of the phosphorylated T-loop to solvent and λ-phosphatase.104,133 Whereas specific mechanisms, either involving unknown proline-directed kinases or unknown CDK7 regulators/adaptors, should be responsible for CDK4 T172phosphorylation, the mechanisms and enzymes involved in its dephosphorylation also remain to be explored. Until now, only


the proline-directed phosphatase calcineurin has been suggested to dephosphorylate cyclin-free CDK4 at mitosis.134,135 In most cancers, especially the most aggressive ones, both p53 and Rb are inactivated. Whereas p53 itself is mutated and defective in more than 50% of cancers, Rb is infrequently mutated and its deregulation occurs at various stages of the pathway that inactivates it by phosphorylation by CDK4/6.31 Rb function is thus potentially reactivatable in most cancers. For this reason, we believe that innovative therapies aiming at reactivating Rb could be most promising, including in cancers associated with p53 mutation. Every protein involved in CDK4 activation leading to Rb inactivation has been found to be oncogenic or antioncogenic. Considering the multistep character of CDK4 activation with none of the steps being sufficient, this intriguingly indicates that only partial deregulation of CDK4 suffices to promote the occurrence of some cancers. As a corollary, the possible good news would be that even partial therapeutic correction of deregulated CDK4 activation might contribute to reequilibrate the cell proliferation control, without being harmful to normal cells. Mechanisms specifically regulating T172phosphorylation of CDK4 will probably prove targets for oncogenic processes. On the other hand the involved protein kinases might be readily druggable therapeutic targets to correct deregulated CDK4 activity. This hope is supported by our preliminary study in glioblastoma cell lines suggesting that CDK4 T172-phosphorylation would be a central node integrating oncogenic signalling cascades.92

pour la Formation à la Recherche dans l’Industrie et l’Agriculture (FRIA). L.B. and K.C., S.P., and P.P.R. are respectively Scientific Research Workers, Postdoctoral Researcher and Senior Research Associate of the FRS-FNRS. References 1. 2.

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13. 14.

Acknowledgements

We thank Dr. Robert P. Fisher for useful comments on our recent study of the differential regulation of CDK4 and CDK6. The continued interest, helpful discussions and moral support by Prof. Jacques Dumont are deeply acknowledged. Work performed in the authors’ laboratory was supported by grants from the Communauté française de Belgique—Actions de Recherches Concertées, the Belgian Fund for Scientific Medical Research (FRSM), the National Fund for Scientific Research (FRS-FNRS, Belgium) and Télévie. X.B. and H.K. are or have been fellows of the Fonds


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