An important role of CDK inhibitor p18(INK4c) in modulating antigen receptor-mediated T cell proliferation.

The inhibitors of cyclin-dependent kinase (CDK) 4 (INK4) bind CDK4/6 to prevent their association with D-cyclins and G(1) cell cycle initiation and progression. We report here that among the seven CDK inhibitors, p18(INK4c) played an important role in modulating TCR-mediated T cell proliferation. Loss of p18(INK4c) in T cells led to hyperproliferation in response to CD3 stimulation. p18(INK4c)-null mice developed lymphoproliferative disorder and T cell lymphomas. Expression of IL-2, IL-2R-alpha, and the major G(1) cell cycle regulatory proteins was not altered in p18-null T cells. Both FK506 and rapamycin efficiently inhibited proliferation of p18-null T cells. In activated T cells, p18(INK4c) remained constant, and preferentially associated with and inhibited CDK6 but not CDK4. We propose that p18(INK4c) sets an inhibitory threshold in T cells and one function of CD28 costimulation is to counteract the p18(INK4c) inhibitory activity on CDK6-cyclin D complexes. The p18(INK4c) protein may provide a novel target to modulate T cell immunity.     

Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.     

Induced expression of p16(INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes

To investigate the mode of action of the p16(INK4a) tumor suppressor protein, we have established U2-OS cells in which the expression of p16(INK4a) can be regulated by addition or removal of isopropyl-beta-D-thiogalactopyranoside. As expected, induction of p16(INK4a) results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16(INK4a) also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27(KIP1). Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16(INK4a), this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16(INK4a). Sequestration of CDK4 by p16(INK4a) allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16(INK4a), p27(KIP1) appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27(KIP1) and p21(CIP1). Significantly, p16(INK4a) itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.     

Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4

The cellular mechanisms regulating intestinal proliferation anddifferentiation remain largely undefined. Previously, we showed anearly induction of the cyclin-dependent kinase (CDK) inhibitor p21^Waf1/Cip1 in Caco-2 cells, ahuman colon cancer line that spontaneously differentiates into a smallbowel phenotype. The purpose of our present study was to assess thetiming of cell cycle arrest in relation to differentiation in Caco-2cells and to examine the mechanisms responsible for CDK inactivation.Caco-2 cells undergo a relativeG₁/S block and cease toproliferate at day3 postconfluency; an increase in theactivity of terminally differentiated brush-border enzymes (sucrase andalkaline phosphatase) was noted at day6 postconfluency. Cell cycle block wasassociated with suppression of both CDK2 and CDK4 activities, which areimportant for G₁/S progression.Treatment of the CDK immune complexes with the detergent deoxycholate(DOC) resulted in restoration of CDK2, but not CDK4, activity atday 3 postconfluency, suggesting the presence of inhibitory protein(s)binding to the cyclin/CDK2 complex at this time point. An increasedbinding of p21^Waf1/Cip1 to CDK2complexes at day3 postconfluency was noted, suggesting a potential role for p21^Waf1/Cip1in CDK2 inactivation; however, immunodepletion ofp21^Waf1/Cip1 from Caco-2 proteinextracts demonstrated thatp21^Waf1/Cip1 is only partiallyresponsible for CDK2 suppression atday 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears tocontribute to the CDK2 inactivation noted atdays6 and12 postconfluency. Taken together, ourresults suggest that multiple mechanisms contribute to CDK suppressionduring Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leadsto G₁ arrest and inhibition ofproliferation that precede Caco-2 cell differentiation.

     

Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.

Cyclin-dependent kinases 4 and 6 are complexed with many small cellular proteins in vivo. We have isolated cDNA sequences, INK4d, encoding a 19-kDa protein that is associated with CDK6 in several hematopoietic cell lines. p19 shares equal similarity and a common ancestor with other identified inhibitors of the p16/INK4 family. p19 interacts with and inhibits the activity of both CDK4 and CDK6 and exhibits no detectable interaction with the other known CDKs. p19 protein is present in both cell nuclei and cytoplasm. The p19 gene has been mapped to chromosome 19p13.2, and the level of its mRNA expression varies widely between different tissues. In contrast to p21 and p27 whose interaction with CDK subunits is dependent on or stimulated by the cyclin subunit, the interaction of p19 and p18 with CDK6 is hindered by the cyclin protein. Binary cyclin D1-p18/p19 or cyclin D1-CDK6 complexes are highly stable and cannot be dissociated by excess amounts of cyclin D1 or p19/p18 proteins, suggesting that p16 inhibitors and D cyclins may interact with CDKs 4 and 6 in a competing or potentially mutually exclusive manner.     

CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms

Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.     

Distinct versus redundant properties among members of the INK4 family of cyclin-dependent kinase inhibitors

p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin-dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis.     

Reducing CDK4/6-p16(INK4a) interface. Computational alanine scanning of a peptide bound to CDK6 protein

The tumor suppressor gene p16INK4a is commonly found altered in numerous and different types of cancer. The encoded protein arrests cell cycle in G1 phase by binding to CDK4 and CDK6, inhibiting their kinase function. In 1995, a 20-residue peptide, extracted from p16INK4a protein sequence, was discovered that retains the cell cycle inhibition properties of the endogenous tumor suppressor. However, its structure has not been determined yet. In this article, the features of a theoretical structure of the peptide bound to CDK6 are reported. The complex was modeled from CDK6-p16INK4a X-ray crystal structure and through molecular dynamics. Final structure was assessed by comparing computed binding free energy changes, when single-alanine substitutions were brought about on the peptide, to experimental data. Better concordance was obtained when including a high level of solvation effects. Solute-solvent vdW energy and electrostatic energy between solute and first shells of water, computed through a force field and considering explicit waters, were also to be included to achieve reasonably good concordance between theoretical and experimental data.     

p27Kip1 acts downstream of N-cadherin-mediated cell adhesion to promote myogenesis beyond cell cycle regulation

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as "community effect".     

The fate of pancreatic tumor cell lines following p16 overexpression depends on the modulation of CDK2 activity

Restitution of lost tumor-suppressor activities may be a promising strategy to target specifically cancer cells. However, the action of ectopically expressed tumor-suppressor genes depends on genetic background of tumoral cells. Ectopic expression of p16(INK4a) induces either cell cycle arrest or apoptosis in different pancreatic cancer cell lines. We examined the molecular mechanisms mediating these two different cellular responses to p16 overexpression. Ectopic expression of p16 leads to G1 arrest in NP-9 cells by redistributing p21/p27 CKIs and inhibiting cyclin-dependent kinase CDK2 activity. In contrast, in NP-18 cells cyclin E (CycE)/CDK2 activity is significantly higher and is not downregulated by p16-mediated redistribution of p21/p27. Moreover, inhibition of CDK4 activity with fascaplysine, which does not affect CycE/CDK2 activity, reduces pocket protein phosphorylation in both cell lines, but fails to induce growth arrest. Like overexpression of p16, fascaplysine induces apoptosis in NP-18 cells, suggesting that inhibition of D-type cyclin/CDK activity in cells with high levels of CycE/CDK2 activity activates an apoptotic pathway. Inhibition of CycE/CDK2 activity via ectopic expression of p21 in NP-18 cells overexpressing p16 induces growth arrest and prevents p16-mediated apoptosis. Accordingly, silencing of p21 expression by using small interfering RNA switches the fate of p16-expressing NP-9 cells from cell cycle arrest to apoptosis. Our data suggest that, after CDK4/6 inactivation, the fate of pancreatic tumor cells depends on the ability to modulate CDK2 activity.     

Lineage specific composition of cyclin D–CDK4/CDK6–p27 complexes reveals distinct functions of CDK4, CDK6 and individual D‐type cyclins in differentiating cells of embryonic origin

Abstract. Objectives: This article is to study the role of G₁/S regulators in differentiation of pluripotent embryonic cells. Materials and methods: We established a P19 embryonal carcinoma cell‐based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G₁/S regulators were analysed with respect to growth and differentiation parameters of the cells. Results and Conclusions: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E–CDK2 but not of cyclin D–CDK4/6–p27 complexes. In an exponentially growing P19 cell population, the cyclin D1–CDK4 complex is detected, which is replaced by cyclin D2/3–CDK4/6–p27 complex following density arrest. During endodermal differentiation kinase‐inactive cyclin D2/D3–CDK4–p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2–CDK4–p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D–CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G₁/S transition‐regulating machinery in early embryonic cells.     

INK4d-deficient mice are fertile despite testicular atrophy

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.     

CAK-independent Activation of CDK6 by a Viral Cyclin

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.     

Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro

Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated "reserve cells" that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin alpha7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation.