Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 21-containing c.yclin kinases exist in oth acnve and lnacnve states Hui Zhang, Gregory J. Hannon, and David Beach Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 USA In normal fibroblasts CDKs exist predominantly in p21/PCNA/cyclin/CDK quaternary complexes, whereas in p53-deficient cells, p21 expression is depressed and the kinases are reduced to a cyclin/CDK binary state, p21 is a universal cyclin kinase inhibitor, but we show here that p21-containing complexes exist in both catalytically active and inactive forms. This finding challenges the current view that active cyclin kinases function only in the binary state and reveals the subtlety with which tumor-suppressor proteins modulate the cell cycle. [Key Words: p21-containing cyclin kinase; cell cycle progression; human fibroblasts; quaternary complex; kinase inhibitor] Received May 12, 1994; revised version accepted June 16, 1994. Much of our current understanding of the regulation of their relative affinities vary with each enzyme (Gu et al. the cell division cycle has emerged from studies of a 1993; Harper et al. 1993; Xiong et al. 1993b). Further- family of protein kinases [cdc2, CDC28, and generically more, several lines of evidence suggest that p21 expres- cyclin-dependent kinase (CDK)] and their inhibitors and sion is regulated by the p53 tumor-suppressor protein activators (for review, see Sherr 1993). A critical step in (E1-Deiry et al. 1993; Xiong et al. 1993b). Thus, cells understanding cell cycle control was the discovery that derived from certain p53-deficient Li-Fraumeni patients CDKs interact with cyclins, proteins that serve as essen- lack p21 associated with cyclin kinases (Xiong et al. tial activating subunits and specificity determinants of 1993a). The p21 gene has a p53 transcriptional regulatory the kinases (Draetta 1990; Sherr 1993). In human cells, motif (E1-Deiry et al. 1993), and cells lacking p53 express multiple cyclins and CDKs interact in a relatively pro- very low levels of p21 (E1-Deiry 1993; Xiong et al. 1993b). miscuous fashion to form a large family of related cyclin These findings have led to a model in which p21 serves kinases, each of which is presumed to play a specific role as an effector of cell cycle arrest in response to activation in cell cycle progression. of the p53 checkpoint pathway (Xiong et al. 1993a). The view that mammalian cyclin kinases exist pre- The concept that p21 is a universal cell cycle kinase dominantly in a binary (cyclin/CDK) state prevailed un- inhibitor creates a paradox, as in normal proliferating til these enzymes were examined in normal human fi- fibroblast cells the majority of multiple kinases exist in broblasts, rather than the many oncogenically trans- quaternary complexes (Zhang et al. 1993). Here, we formed cell types that had been investigated hitherto present the unexpected finding that p21 is a component (Xiong et al. 1992). In normal fibroblasts the major pop- of catalytically active cyclin kinases. It appears that p21- ulation of multiple cyclin kinases exists in quatemary containing enzymes can transition between active and complexes consisting of cyclin, CDK, proliferating cell inactive states, probably through changes in the stoichi- nuclear antigen (PCNA), and a protein of apparent Mr ometry of the p21 subunit. These findings have broad 21,000, p21 (Zhang et al. 1993). However, in fibroblasts implications for our understanding of the normal cell that are transformed with a variety of tumor viral onco- cycle and the subversion of control pathways in tumor proteins the quatemary complexes essentially reduce to cells. a binary state. Deregulation of cell proliferation is a hall- mark of neoplastic transformation. Difference in the Results composition of cell cycle kinases between normal and p21 associates with PCNA and multiple cyclin kinases transformed cells suggests that fundamental changes in cell cycle regulation contribute to neoplastic transforma- We have demonstrated previously the association of p21 tion. with multiple cyclin kinase complexes. To examine the Recently, it has become apparent that p21 is a univer- full spectrum of p21-associated proteins, we raised a spe- sal inhibitor of cyclin/CDK catalytic activity (Gu et al. cific p21 antiserum. This serum immunoprecipitated 1993; Harper et al. 1993; Xiong et al. 1993b). Each mem- from lysates of 3SS-labeled normal human diploid fibro- ber of the cyclin kinase family is inhibited by p21, but blasts (WI38), a prominent -21-kD protein, and a hum- 1750 GENES& DEVELOPMENT8:1750-1758 91994 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/94 $5.00 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press p21-containing cyclin kinases bet of additional polypeptides {Fig. 1A, anti-p21). Partial ular weight than the cyclin A-containing complexes. V8 protease digestion revealed identity between the This suggests the presence of an additional cyclin D-as- -21-kD band and the p21 protein derived from in vitro sociated subunit, possibly the -105-kD protein that translation of the p21 cDNA (Fig. 1B). To aid in the iden- cosediments with these complexes (Fig. 1C). Our results tification of the proteins that coimmunoprecipitate with are also consistent with the existence of an independent p21, immunoprecipitates of CDC2, CDK2, CDK4, cyclin interaction between p21 and PCNA. A, cyclin B1 and cyclin D1 were electrophoresed in par- allel. By comparison, the mobilities of most of the p21- associated proteins could be correlated with that of cy- Active and inactive forms of the p21-associated clins, CDKs, and PCNA, as described earlier (see Fig 1A; cyclin kinases in vitro Xiong et al. 1993a; Zhang et al. 1993). These results sug- gest that the cyclin kinases and PCNA are the major p21 is a universal inhibitor of cyclin kinases in vitro (Gu p21-associated proteins in WI38 cells. To determine et al. 1993; Harper et al. 1993; Xiong et al. 1993b). Thus, whether the majority of cellular p21 exists in complexes it was predicted that all p21-containing complexes with these proteins, we fractionated extracts of 3SS-la- would lack catalytic activity. However, in previous in beled WI38 cells by sedimentation through a glycerol vitro experiments, progressive addition of p21 to cyclin gradient and examined p21 immunoprecipitates from kinases did not result in a precise, corresponding loss of each fraction (Fig. 1C). Little or no monomeric p21 was kinase activity but, instead, caused an abrupt transition evident. The majority of p21 cosedimented with cyclin from full activity to essentially complete inhibition kinase complexes, particularly those that appear to con- (Xiong et al. 1993b). Here, we have examined the seem- tain cyclin A/CDK2 and cyclin D1/CDK4. The former ing cooperativity of p21 inhibition of cyclin kinases in probably comprise cyclin A/CDK2/p21/PCNA quater- greater detail. nary complexes. We noted that in this gradient, cyclin Lysates were prepared from metabolically 3SS-labeled Dl-containing complexes had a higher apparent molec- insect cells infected with baculoviruses directing the ex- Figure 1. PCNA and cyclin/kinases are the major p21 associated proteins. (A) p21-associated proteins were immunoprecipitated from cell lysates of 35S-labeled normal human fibroblasts (WI38) and compared with the proteins immunoprecipitated by anti-CDC2, CDK2, CDK4, cyclin A, cyclin B, or cyclin D 1 antisera as indicated. The positions of protein molecular mass markers (in kD), electrophoresed in parallel, are indicated. (B} p21 produced by in vitro trans- lation of the p21 cDNA (left) is compared with p21 purified from anti-p21 immunoprecipitates from 3SS-labeled human cell lysates by partial V8 protease digestion (right). (C) Glycerol gradient sedimentation analysis of p21-associated protein complexes. The fractions were taken from top (left) to bottom (right) of the gradient and immunoprecipitated with anti-p21 antibody. The peak of BSA, sedimented in parallel, migrates at fraction 16. GENES & DEVELOPMENT 1751 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Zhang et al. pression of human cyclin A, CDK2, and p21. Progressive p21 that was recovered with Ni +-NTA agarose (data not addition of the p21-containing lysate to preformed cyclin shown). These results demonstrate that p21-containing A/CDK2 complexes resulted in accumulation of p21/ cyclin kinase complexes can exist in both active and cyclin A/CDK2 ternary complexes (Fig. 2A). At a spe- inactive states. Inhibition is achieved only as p21 cific point in the titration, the histone H1 kinase activity reaches a saturating concentration. in cyclin A immunoprecipitates was abruptly lost (Fig. We note that p2! displays an altered electrophoretic 2A). As noted previously, this transition between active mobility when added to cyclin kinases at noninhibitory and inactive enzyme was not reflected directly in the levels. Several lines of evidence indicate that altered mo- binding of p21 to cyclin A/CDK2, which was approxi- bility reflects p21 phosphorylations. These are discussed mately proportional to the amount of p21 added (Fig. in detail below. 2A). One explanation for the observed discrepancy be- p21 is a universal inhibitor of cyclin kinases, and non- tween p21 binding and inhibition is that p21 might fail linear p21 inhibition profiles have been observed with to act as a cyclin kinase inhibitor at subsaturating levels. cyclin A/CDK2, cyclin E/CDK2, and cyclin D1/CDK4 To test this possibility, we specifically recovered the enzymes (Xiong et al. 1993b; Fig. 3), suggesting that the p21-containing complexes using the p21 antiserum (Fig. association of p21 with active cyclin kinases might be a 2B). Contrary to previous predictions, complexes formed general phenomenon. Consistent with this prediction, at subsaturating p21 concentrations possessed substan- we detected substantial kinase activity in p21/cyclin tial histone H 1 kinase activity.