Identification of Human and Mouse p19, a Novel CDK4 and CDK6 Inhibitor with Homology to p16^ink4

Abstract

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G 1 /S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G 1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G 1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G 1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.Apoptosis in immature T cells and T-cell hybridomas, which may relate to negative selection during T-cell development, can be initiated by signals through the T-cell receptor-CD3 complex (18,32,33). This process of activation-induced apoptosis (anti-CD3 apoptosis) consists of two distinct phases. The first phase is the cell cycle block at the G 1 /S transition; it is followed by the second phase, with the generation of apoptotic DNA ladders (18). The second phase requires extracellular calcium and can be inhibited by the immunosuppressive drug cyclosporin A (18). We and others have shown that Nur77 (NGFI-B) orphan steroid receptor is induced during anti-CD3 apoptosis through the calcium signals and it plays an essential role in the cell death process (17, 35). Dominant negative Nur77 can block apoptosis but not the interleukin 2 production of anti-CD3-treated T-cell hybridomas (35). Thus, Nur77 is involved in the second phase of anti-CD3 T-cell apoptosis.As alluded to above, anti-CD3 death in T-cell hybridomas is also accompanied by a G 1 cell cycle block. In all organisms studied so far, cell cycle progression is mediated by cyclindependent kinases (CDKs) that consist of a catalytic subunit CDK and a regulatory subunit cyclin. In mammalian cells, cyclin E-CDK2 together with cyclin D-CDK4 and/or cyclin D-CDK6, which are active in the G 1 phase, control the G 1 -to-S transition (23, 24, 28, 31 and references therein). There are at least three different D-type cyclins, with T cells expressing cyclins D2 and D3 and two cyclin D-associating kinases, CDK4 and CDK6. One of their substrates is the retinobl...

The Nuclear Protein p34SEI-1 Regulates the Kinase Activity of Cyclin-Dependent Kinase 4 in a Concentration-Dependent Manner

“…Under our in vitro assay conditions, p34 SEI-1 acts as both an activator and inhibitor of CDK4, which distinguishes p34 SEI-1 from other CDK4-binding proteins. INK4 (5)(6)(7)(8)(9)(10)(11) and KIP proteins (12)(13)(14) only inhibit CDK4; gankyrin functions as a competitor to antagonize INK4 proteins (17), while Tax is competent in activating CDK4 (15,16), quenching p16 (32,33), and affecting cyclin Ds (34). It is important to point out that most of these studies were performed by in vitro or cell-based studies, and their biological relevance remains to be established.…”

“…Many proteins have been found to act as negative or positive kinase regulators of CDK4 and 6 through protein/protein interactions. While binding of D cyclins is required for the full kinase activity (4), INK4 (including p16, p15, p18, and p19) (6)(7)(8)(9)(10)(11) and KIP (including p21, p27, and p57) proteins (5,(12)(13)(14) are inhibitors of CDK4 and 6. Moreover, the oncoprotein Tax from human T-cell leukemia virus 1 (HTLV-1) stimulates the CDK4 activity in infected cells through physical association (15,16), and the oncoprotein gankyrin affects CDK4 by counteracting against the inhibition of p16 and p18 (17).…”

INK4 cell cycle inhibitors direct transcriptional inactivation of NF-κB

“…Signi®cant amounts of INK4 molecules occur as free molecules in human cell lines (Della Ragione et al, 1996), thus it is possible that they have other targets than the formally known Cdk4/Cdk6. Suggestions about their cellular role is given by Chan et al (1995) who showed that p19INK4 interacts with the apoptosis inducing orphan steroid receptor Nur77. Furthermore, the yeast Cdk inhibitor Pho81 associates with the transcription factor Pho4, thereby preventing its ability to activate transcription (Hirst et al, 1994).…”

“…In this scenario, it was shown that p19INK4 interacts with Nur77 (Chan et al, 1995) and yeast Pho81 (containing high similarity to mammalian p16INK4) interacts with Pho4 (Hirst et al, 1994), proteins which are totally unrelated to Cdks. Our results indicate that NF-kBp65 could be a target of INK4 proteins, which interact physically with NF- 3 and 4).…”

Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

“…Evidence of p16s role as a tumour suppressor protein also comes from observations that families carrying p16 mutations are predisposed for malignant melanomas (Hussusain et al, 1994;Ranade et al, 1995) and that mice lacking p16 develop tumours early . P16 INK4a/CDKN2A (Serrano et al, 1994) and its closely related INK4/CDKN2 family members p15 INK4b (p15) , p18 INK4c (p18) (Guan et al, 1994;Hirai et al, 1995) and p19 INK4d (p19) (Hirai et al, 1995;Chan et al, 1995) interact speci®cally with cdk4 and cdk6 and prevent the phosphorylation of pRb by the cyclin D-CDK complex in vitro. However, only p16 is strongly linked to tumour suppression activity.…”