Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. The crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) is reported at 2.25 Å resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates (Honda, 2005).
In many organisms, initiation and progression through the G1 phase of the cell cycle requires the activity of G1-specific cyclins (cyclin D and cyclin E) and their associated cyclin-dependent kinases (CDK2, CDK4, CDK6). The C. elegans genes cyd-1 and cdk-4, encoding proteins similar to cyclin D and its cognate cyclin-dependent kinase, respectively, are necessary for proper division of postembryonic blast cells. Animals deficient for cyd-1 and/or cdk-4 activity have behavioral and developmental defects that result from the inability of the postembryonic blast cells to escape G1 cell cycle arrest. Moreover, ectopic expression of cyd-1 and cdk-4 in transgenic animals is sufficient to activate an S-phase reporter gene. No embryonic defects associated with depletion of either of these two gene products is observed, suggesting that their essential functions are restricted to postembryonic development. It is proposed that the cyd-1 and cdk-4 gene products are an integral part of the developmental control of larval cell proliferation through the regulation of G1 progression (Park, 1999).
To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, the mouse Cdk4 locus has been targeted by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types (Rane, 1999).
The mechanism by which cyclin-dependent kinase 4 (CDK4) regulates cell cycle progression is not entirely clear. Cyclin D/CDK4 appears to initiate phosphorylation of retinoblastoma protein (Rb) leading to inactivation of the S-phase-inhibitory action of Rb. However, cyclin D/CDK4 has been postulated to act in a noncatalytic manner to regulate the cyclin E/CDK2-inhibitory activity of p27(Kip1) by sequestration. The roles of CDK4 in cell cycle regulation have been investigated by targeted disruption of the mouse CDK4 gene. CDK4(-/-) mice survive embryogenesis and show growth retardation and reproductive dysfunction associated with hypoplastic seminiferous tubules in the testis and perturbed corpus luteum formation in the ovary. These phenotypes appear to be the opposite of those for p27-deficient mice, such as gigantism and gonadal hyperplasia. A majority of CDK4(-/-) mice develop diabetes mellitus by 6 weeks, associated with degeneration of pancreatic islets. Fibroblasts from CDK4(-/-) mouse embryos proliferate similarly to wild-type embryonic fibroblasts under conditions that promote continuous growth. However, quiescent CDK4(-/-) fibroblasts exhibit a substantial (approximately 6-h) delay in S-phase entry after serum stimulation. This cell cycle perturbation by CDK4 disruption is associated with increased binding of p27 to cyclin E/CDK2 and diminished activation of CDK2 accompanied by impaired Rb phosphorylation. Importantly, fibroblasts from CDK4(-/-) p27(-/-) embryos display partially restored kinetics of the G0-S transition, indicating the significance of the sequestration of p27 by CDK4. These results suggest that at least part of CDK4's participation in the rate-limiting mechanism for the G0-S transition consists of controlling p27 activity (Tsutsui, 1999).
Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of a linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. When it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhances their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase (Diehl, 1997).
Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Cak1p and p40(MO15) have strikingly different substrate specificities. Cak1p preferentially phosphorylates monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40(MO15) only phosphorylates cdk6 bound to cyclin D3, whereas Cak1p recognizes monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. cdk inhibitors, including p21(CIP1), p27(KIP1), p57(KIP2), p16(INK4a), and p18(INK4c), can block phosphorylation by p40(MO15) but not phosphorylation by Cak1p. These results demonstrate that although both Cak1p and p40(MO15) activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly (Kaldis, 1998).
The cdk-activating kinase (CAK) activates cyclin-dependent kinases (cdks) that control cell-cycle progression by phosphorylating a threonine residue conserved in cdks. CAK from humans contains p40MO15 (cdk7), cyclin H and MAT1, which are also subunits of transcription factor IIH where they phosphorylate the C-terminal domain of the large subunit of RNA polymerase II. In contrast, budding yeast Cak1p is a monomeric enzyme without C-terminal domain kinase activity. CAK activities have been analyzed in HeLa cells using cdk2-affinity chromatography. In addition to MO15, a second CAK activity was detected that runs on gel filtration at 30-40 kDa. This activity phosphorylates and activates cdk2 and cdk6. Furthermore, this 'small CAK' activity resembled Cak1p rather than MO15 in terms of substrate specificity, reactivity to antibodies against MO15 and Cak1p, and sensitivity to 5'-fluorosulfonylbenzoyladenosine, an irreversible inhibitory ATP analog. These findings suggest the presence of at least two different CAK activities in human cells (Kaldis, 2000).
A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activates extracellular signal-regulated protein kinases (ERKs) and induces expression of cyclin D1. Newly synthesized cyclin D1 assembles with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that inefficiently phosphorylate the retinoblastoma protein. Activation of the MEK1/ERK pathway triggers neither degradation of the CDK inhibitor kinase inhibitory protein-1 (p27Kip1) nor leads to activation of cyclin E- and A-dependent CDK2, and such cells do not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits, generates levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27Kip1 is mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes are activated, and serum-starved cells enter S phase. Thus, although the activity of p27Kip1 normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequester p27Kip1 and reduce the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells complete S phase and divide, but they are unable to continuously proliferate, indicating that other serum-responsive factors ultimately become rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27Kip1-mediated inhibition (Cheng, 1998).
The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), it has been shown that this kinase plays a significant role in positively regulating the expression of cyclin D1. This is found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells (Ladha, 1998).
Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). The cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27(kip1) but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Transforming growth factor beta1 (TGFbeta1) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH do not interfere importantly with TGFbeta receptor signaling, because they did not affect the TGFbeta-induced nuclear translocation of Smad 2 and 3. TGFbeta inhibits the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFbeta does not inhibit c-myc expression. In TSH-stimulated cells, TGFbeta does not affect the expression of cyclin D3, cdk4, and p27(kip1), nor the induced formation of cyclin D3-cdk4 complexes, but it prevents the TSH-induced relocalization of p27(kip1) from cdk2 to cyclin D3-cdk4. It prevents the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they are prevented from sequestering nuclear p27(kip1) away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27(kip1). It provides a new mechanism for regulation of proliferation by TGFbeta, which points out the subcellular location of cyclin D-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture (Depoortere, 2000).
The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, the cyclin-dependent kinase 4 (CDK4) gene has been identified as a transcriptional target of c-MYC. c-MYC induces a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay is associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviates the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation (Hermeking, 2000).
Because a temporal arrest in the G(1) phase of the cell cycle is thought to be a prerequisite for cell differentiation, cell cycle factors were investigated that critically influence the differentiation of mouse osteoblastic MC3T3-E1 cells induced by bone morphogenetic protein 2 (BMP-2), a potent inducer of osteoblast differentiation. Of the G(1) cell cycle factors examined, the expression of cyclin-dependent kinase 6 (Cdk6) was found to be strongly down-regulated by BMP-2/Smads signaling, mainly via transcriptional repression. The enforced expression of Cdk6 blocked BMP-2-induced osteoblast differentiation to various degrees, depending on the level of its overexpression. However, neither BMP-2 treatment nor Cdk6 overexpression significantly affected cell proliferation, suggesting that the inhibitory effect of Cdk6 on cell differentiation was exerted by a mechanism that is largely independent of its cell cycle regulation. These results indicate that Cdk6 is a critical regulator of BMP-2-induced osteoblast differentiation and that its Smads-mediated down-regulation is essential for efficient osteoblast differentiation (Ogasawara, 2004).
Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme (Kato, 1994).
The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. The CDK inhibitors p21CIP, p27KIP, and p57KIP2 all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase association constant, respectively, mostly because of a decrease in disassociation constant. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, the assembly of endogenous complexes was examined in mammary epithelial cells after release from a G0 arrest. Cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. All three CIP/KIP inhibitors are found to target cdk4 and cyclin D1 to the nucleus. It has been suggested that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions. Although all three of the inhibitors behave similarly with respect to promoting complex assembly, only p21 promotes the assembly of complexes with active RB kinase activity. Under the same conditions as those used for p21, both p27 and p57 cause only a gradual loss of cdk4-associated RB kinase activity. Possibly p27 and p57 may act only as inhibitors, revealing some fundamental difference between the way they interact with cdk/cyclin D complexes and the way p21 interacts (LaBaer, 1997).
Although p27(Kip1) has been considered a general inhibitor of G1 and S phase cyclin-dependent kinases, the interaction of p27 with two such kinases, cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu cells containing a p27 inducible system, a 6-fold increase over the basal p27 level completely inhibits Cdk2 and cell cycle progression. In contrast, the same or a larger increase in p27 levels does not inhibit Cdk4 or its homolog Cdk6, despite extensive binding to these kinases. A p27-cyclin A-Cdk2 complex formed in vitro is essentially inactive, whereas a p27-cyclin D2-Cdk4 complex is active as a retinoblastoma kinase and serves as a substrate for the Cdk-activating kinase Cak. High concentrations of p27 inhibit cyclin D2-Cdk4, apparently through the conversion of active complexes into inactive ones by the binding of additional p27 molecules. In contrast to their differential interaction, cyclin A-Cdk2 and cyclin D2-Cdk4 are similarly inhibited by bound p21(Cip1/Waf1). The role of cyclin A-Cdk2 as a p27 target, and the role of cyclin D2-Cdk4 as a p27 reservoir may result from the differential ability of bound p27 to inhibit the kinase subunit in these complexes (Blain, 1997).
Loss-of-function mutations of p16(INK4a) have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro; this inhibition is believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4(N158), designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, is unable to arrest the cell cycle of the same cells. Functional differences have now been determined between p16 and Cdk4(N158). p16 and Cdk4(N158) inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociates cyclin D1-Cdk4 complexes with the release of bound p27(KIP1), while Cdk4(N158) forms complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 forms complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities are correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E become resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression (Jiang, 1998).
Cyclin D-Cdk4 complexes have a demonstrated role in G1 phase, regulating the function of the retinoblastoma susceptibility gene product (Rb). Following treatment with low doses of UV radiation, cell lines that express wild-type p16 and Cdk4 respond with a G2 phase cell cycle delay. The UV-responsive lines contain elevated levels of p16 post-treatment, and the accumulation of p16 correlates with the G2 delay. In UV-irradiated HeLa and A2058 cells, p16 binds Cdk4 and Cdk6 complexes with increased avidity and inhibits a cyclin D3-Cdk4 complex normally activated in late S/early G2 phase. Activation of this complex is correlated with the caffeine-induced release from the UV-induced G2 delay and a decrease in the level of p16 bound to Cdk4. Finally, overexpression of a dominant-negative mutant of Cdk4 blocks cells in G2 phase. These data indicate that the cyclin D3-Cdk4 activity is necessary for cell cycle progression through G2 phase into mitosis and that the increased binding of p16 blocks this activity and G2 phase progression after UV exposure (Gabrielli, 1999).
The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D-CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity is well tolerated and has no overt effects on the cell cycle (Cheng, 1999).
The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules (Parry, 1999).
The cyclin-dependent kinases 4 and 6 (Cdk4/6) that drive progression through the G1 phase of the cell cycle play a central role in the control of cell proliferation, and CDK deregulation is a frequent event in cancer. Cdk4/6 are regulated by the D-type cyclins, which bind to CDKs and activate the kinase, and by the INK4 family of inhibitors. INK4 proteins can bind both monomeric CDK, preventing its association with a cyclin, and also the CDK-cyclin complex, forming an inactive ternary complex. In vivo, binary INK4-Cdk4/6 complexes are more abundant than ternary INK4-Cdk4/6-cyclinD complexes, and it has been suggested that INK4 binding may lead to the eventual dissociation of the cyclin. The 2.9-Å crystal structure of the inactive ternary complex between Cdk6, the INK4 inhibitor p18INK4c, and a D-type viral cyclin is presented here. The structure reveals that p18INK4c inhibits the CDK-cyclin complex by distorting the ATP binding site and misaligning catalytic residues. p18INK4c also distorts the cyclin-binding site, with the cyclin remaining bound at an interface that is substantially reduced in size. These observations support the model that INK4 binding weakens the cyclin's affinity for the CDK. This structure also provides insights into the specificity of the D-type cyclins for Cdk4/6 (Jeffrey, 2000).
The product (pRb) of the retinoblastoma gene (RB-1) prevents S-phase entry during the cell cycle, and inactivation of this growth-suppressive function is presumed to result from pRb hyperphosphorylation during late G1 phase. Complexes of the cyclin-dependent kinase, cdk4, and each of three different D-type cyclins, assembled in insect Sf9 cells, phosphorylate a pRb fusion protein in vitro at sites identical to those phosphorylated in human T cells. Only D-type cyclins activate cdk4 enzyme activity, whereas cyclins A, B1, and E do not. When Sf9 cells were coinfected with baculovirus vectors encoding human pRb and murine D-type cyclins, cyclins D2 and D3, but not D1, bind pRb with high stoichiometry in intact cells. Introduction of a vector encoding cdk4, together with those expressing pRb and D-type cyclins, induces pRb hyperphosphorylation and dissociation of cyclins D2 and D3, whereas expression of a kinase-defective cdk4 mutant in lieu of the wild-type catalytic subunit yields ternary complexes. The transcription factor E2F-1 also binds to pRb in insect cells. Coexpression of cyclin D-cdk4 complexes, but to neither subunit alone triggers pRb phosphorylation and prevents its interaction with E2F-1. The D-type cyclins may play dual roles, both as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates (Kato, 1993).
Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. The consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2. Cyclin D1-Cdk4 efficiently phosphorylates the G1 peptide, RPPTLS780PIPHIPR that contains a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 do not. To determine the phosphorylation state of pRB in vitro and in vivo, a specific antibody was raised against phospho-Ser780 in pRB. Cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylates Ser780 in recombinant pRB. The Ser780 in pRB is phosphorylated in the G1 phase in a cell cycle-dependent manner. Furthermore, pRB phosphorylated at Ser780 cannot binds to E2F-1 in vivo. These data show that cyclin D1-Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo (Kitagawa, 1996).
The retinoblastoma protein (pRb) inhibits progression through the cell cycle. Although pRb is phosphorylated when G1 cyclin-dependent kinases (Cdks) are active, the mechanisms underlying pRb regulation are unknown. In vitro phosphorylation by cyclin D1/Cdk4 leads to inactivation of pRb in a microinjection-based in vivo cell cycle assay. In contrast, phosphorylation of pRb by Cdk2 or Cdk3 in complexes with A- or E-type cyclins is not sufficient to inactivate pRb function in this assay, despite extensive phosphorylation and conversion to a slowly migrating 'hyperphosphorylated form'. The differential effects of phosphorylation on pRb function coincide with modification of distinct sets of sites. Serine 795 is phosphorylated efficiently by Cdk4, even in the absence of an intact LXCXE motif in cyclin D, but not by Cdk2 or Cdk3. Mutation of serine 795 to alanine prevents pRb inactivation by Cdk4 phosphorylation in the microinjection assay. This study identifies a residue whose phosphorylation is critical for inactivation of pRb-mediated growth suppression, and it indicates that hyperphosphorylation and inactivation of pRb are not necessarily synonymous (Connell-Crowley, 1997).
Evidence is presented that phosphorylation of the C-terminal region of Rb by Cdk4/6 initiates successive intramolecular interactions between the C-terminal region and the central pocket. The initial interaction displaces histone deacetylase from the pocket, blocking active transcriptional repression by Rb. This facilitates a second interaction that leads to phosphorylation of the pocket by Cdk2 and disruption of pocket structure. These intramolecular interactions provide a molecular basis for sequential phosphorylation of Rb by Cdk4/6 and Cdk2. Cdk4/6 is activated early in G1, blocking active repression by Rb. However, it is not until near the end of G1, when cyclin E is expressed and Cdk2 is activated, that Rb is prevented from binding and inactivating E2F (Harbour, 1999).
MyoD has been proposed to facilitate terminal myoblast differentiation by binding to and inhibiting phosphorylation of the retinoblastoma protein (pRb). MyoD can interact with cyclin-dependent kinase 4 (cdk4) through a conserved 15 amino acid (aa) domain in the C-terminus of MyoD. MyoD, its C-terminus lacking the basic helix-loop-helix (bHLH) domain, or the 15 aa cdk4-binding domain all inhibit the cdk4-dependent phosphorylation of pRb in vitro. Cellular expression of full-length MyoD or fusion proteins containing either the C-terminus or just the 15 aa cdk4-binding domain of MyoD inhibits cell growth and pRb phosphorylation in vivo. The minimal cdk4-binding domain of MyoD fused to GFP can also induce differentiation of C2C12 muscle cells in growth medium. The defective myogenic phenotype in MyoD-negative BC3H1 cells can be rescued completely only when MyoD contains the cdk4-binding domain. It is proposed that a regulatory checkpoint in the terminal cell cycle arrest of the myoblast during differentiation involves the modulation of the cyclin D cdk-dependent phosphorylation of pRb through the opposing effects of cyclin D1 and MyoD. Attempts to detect interaction between MyoD or myogenin and pRb in the two-hybrid assay were unsuccessful. In contrast to the suggestion from in vitro immunoprecipitation studies, neither MyoD nor myogenin were found to interact with pRb. Thus, if there is any direct interaction between pRb and the myogenic factors in C2C12 cells it appears to be weak. In support of these observations, early targets of MyoD transcription are fully induced in cells lacking pRb, making the significance of the in vitro MyoD-pRb interaction questionable. Preliminary experiments indicate that the MyoD homologs from Drosophila (Nautilus) and Caenorhabditis elegans (hlh-1) can specifically bind vertebrate cdk4 and can inhibit cell growth in the BrdU incorporation assay. Virtually all cyclin D1-dependent kinase activity in proliferating mouse fibroblasts can be attributed to cdk4; cyclin D1 is the only ectopically expressed cyclin that will inhibit myogenesis, consistent with a unique role for the MyoD-cdk4 interaction. These results, however, do not rule out a similar interaction with cdk6, since binding between the 15 aa MyoD cdk4-binding site and cdk6 has also been observed (Zhang, 1999).
The dynamics of the MyoD-cdk4 interaction in the myoblast can be represented by the following model. The forced overexpression of MyoD inhibits the cdk4-dependent phosphorylation of pRb to trigger growth arrest and the exit from the cell cycle (likely to include cdk6 as well). Dephosphorylated pRb is thought to be required to maintain cell cycle arrest by inhibiting the growth promoting actions of E2F/DP family members, the induction of apoptosis, and DNA replication in myotubes. Excess cyclin D1 activates more cdk4 and the complex is translocated to the nucleus where it inhibits MyoD and the activation of the myogenic program. Excess expression of nuclear cdk4 triggers phosphorylation of pRb, allowing dissociation of E2F/DP and cell growth. In contrast, excess ectopic expression of MyoD depletes active cdk4, prevents the phosphorylation of pRb and induces growth arrest while activating target genes to drive myogenesis. Thus, excess cyclin D1 and cdk4 induce growth and block differentiation, whereas excess MyoD induces exit from the cell cycle and differentiation. D-type cyclins act as growth factor sensors and levels of cyclin D1 appear to be rate limiting in the formation of active cdk4, based upon the half-life of each protein. The ectopic expression of cyclin D1 can increase nuclear levels of cdk4 to shorten the G1 phase of the cell cycle. Thus, the relative nuclear ratios of MyoD and cyclin D cdks in the cell would appear to be key determinants in the cell cycle decisions of the myoblast during terminal differentiation, and this ratio is determined by growth factor modulation of cyclin D1 expression levels and the cyclin D1-dependent translocation of active cdk4 to the nucleus (Zhang, 1999 and references therein).
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