Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis

Key Points A Cdk6 R31C knock-in mutation resistant to INK4 inhibitors cooperates with Cdk4 hyperactivity in the development of hematopoietic tumors. In Cdk6 R31C cells, p16INK4a increasingly binds and inhibits Cdk4, suggesting that both kinases cooperate in sequestering INK4 proteins in cancer.

CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16INK4a-Independent Mechanisms

ABSTRACT Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16INK4a and p21CIP1. 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 p16INK4a 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 p16INK4a. 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 p16INK4a. 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 p16INK4a deficiency, CDK4 expression, and p53 or p21CIP1 ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.

The influence of INK4 proteins on growth and self-renewal kinetics of hematopoietic progenitor cells

This study investigated the influence of expression of proteins of the INK4 family, particularly p16, on the growth and self-renewal kinetics of hematopoietic cells. First, retrovirus-mediated gene transfer (RMGT) was used to restore p16INK4aexpression in the p16INK4a-deficient lymphoid and myeloid cell lines BV173 and K562, and it was confirmed that this inhibited their growth. Second, to sequester p16INK4a and related INK4 proteins, cyclin-dependent kinase 4 (CDK4) was retrovirally transduced into normal human CD34+ bone marrow cells and then cultured in myeloid colony-forming cell (CFC) assays. The growth of CDK4-transduced colonies was more rapid; the cell-doubling time was reduced; and, upon replating, the colonies produced greater yields of secondary colonies than mock-untransduced controls. Third, colony formation was compared by marrow cells fromp16INK4a−/− mice and wild-type mice. The results from p16INK4a−/−marrow were similar to those from CDK4-transduced human CFCs, in terms of growth rate and replating ability, and were partially reversed by RMGT ofp16INK4a. Lines of immature granulocytic cells were raised from 15 individual colonies grown from the marrow ofp16INK4a−/−mice. These had a high colony-forming ability (15%) and replating efficiency (96.7%). The p16INK4a−/−cell lines readily became growth factor–independent upon cytokine deprivation. Taken together, these results demonstrate that loss of INK4 proteins, in particular p16INK4a, increases the growth rate of myeloid colonies in vitro and, more importantly, confers an increased ability for clonal expansion on hematopoietic progenitor cells.

Cyclin D-CDK Subunit Arrangement Is Dependent on the Availability of Competing INK4 and p21 Class Inhibitors

ABSTRACT 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. Here we show that although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16INK4a 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 p21CIP1 and p27KIP1 in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21CIP1- or p27KIP1-bound states. In agreement with this hypothesis, overexpression of p21CIP1 in 293 cells, where CDK4 is bound to p16INK4a, stimulates the formation of ternary cyclin D-CDK4-p21CIP1 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.

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.