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. In this report we demonstrate that the CDK inhibitors pZl'^^'', p27^^^, and p57^^^^ 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 in K^, respectively, mostly because of a decrease in X^ff. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a GQ arrest. In agreement with our other data, cyclin Dl and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin Dl complexes. This complex assembly occurs in parallel to an increase in cyclin Dl-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin Dl-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin Dl to the nucleus. We suggest 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.

Genomic Southern blots of DNA from eight strains of mice were examined for restriction fragment length polymorphisms in their loci encoding the variable region of the T-cell receptor beta chain (V beta), using 16 different V beta-specific probes. Mouse strains BALB/c, C57BL/6, C3H, and PL were identical, while strains SJL, C57BR, C57L, and SWR shared several polymorphisms with respect to the other four strains. In addition, SJL, C57L, C57BR, and SWR DNAs were missing 50% of the hybridizing bands visualized in BALB/c DNA. A cDNA library from concanavalin A-stimulated SJL spleen blasts was constructed and examined for V beta gene usage. Ten genes were found to account for all V beta-containing clones isolated, including three newly identified V beta genes. All 10 of these genes were found to be present in BALB/c mice. We conclude that SJL, C57L, C57BR, and SWR mice represent V beta deletion mutants of the BALB/c genotype.

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. Here we report that Cak1p and p40MO15 have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40 MO15 preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40MO15 only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21 CIP1 , p27 KIP1 , p57 KIP2 , p16 INK4a, and p18 INK4c, could block phosphorylation by p40 MO15 but not phosphorylation by Cak1p. Our 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. Structural and physiological implications of these findings will be discussed.

Loss-of-function mutations of p16INK4a 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, and this inhibition is believed to be the cause of the p16-mediated G 1 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, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050-2054, 1993). In this study, we determined functional differences between p16 and Cdk4 N158. We show that p16 and Cdk4 N158 inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27 KIP1 , while Cdk4 N158 formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G 1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.