Cyclin-Dependent Kinase Proteins

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Cyclin-Dependent Kinase Proteins

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Cyclin-Dependent Kinase Proteins Background

Because cyclin accumulation was found to promote its own destruction, this provided a model of mitotic control in which: 1) cyclin activates MPF to drive cells into mitosis; 2) MPF activation promotes cyclin degradation; and 3) cyclin levels fall, thereby leading to MPF inactivation and mitotic exit. The exact function of cyclin was unknown until it was shown to form a heterodimeric complex with Cdc2 leading to kinase activation. As cyclin binding was found to be required for activation of other p34cdc2 homologs, these kinases were renamed cyclin-dependent kinases (CDKs; N.B. as per current convention, the catalytic component of MPF and the gene products of cdc2+ and CDC28 will hereafter be referred to as Cdk1).

Periodically expressed cyclins also form complexes with CDKs to regulate early cell cycle events in yeast and metazoans. The sequential expression of cyclins is controlled by specific, cell cycle-regulated transcription factors. For example, cyclins are absent in G0 (quiescence) and early G1 in metazoans. Growth factor signaling is necessary to transit from G0/G1 into S phase, and signaling through this pathway leads to expression of cyclin D but not any other cyclin. Cyclin D-dependent kinase activity collaborates with the transcription factor E2F to promote the sequential expression of cyclins E, A, and B. These cyclins are also degraded in sequential manner, thus restricting the window of time in which they act. Therefore, a general model for metazoan cell cycle control is that cyclin D regulatesG1, cyclin E controls G1 through S phase, cyclin A regulates the G1/S transition through G2/M, and cyclin B controls G2/M and mitosis. In contrast to budding and fission yeasts in which a single CDK controls both early and late cell cycle events, metazoans have multiple CDK catalytic subunits that become active at different times due to specificity in cyclin pairing: Cdk4 and 6 bind cyclin D, Cdk2 forms complexes with cyclins E and A, and Cdk1 binds cyclins A and B. In both yeast and metazoans, nonetheless, activation of CDKs by cyclins is important for control of two different cell cycle transitions.

Although cyclin binding is necessary for CDK activation, it is generally not sufficient. To become fully active CDKs must also be phosphorylated on a conserved threonine residue in the activation (T-) loop. The importance of this residue was first suggested on the basis of sequence homology to known sites of phosphorylation in Src and Protein Kinase A, and mutation of the predicted phosphorylation site (T167 in S. pombe Cdk1, T161 in human and Xenopus Cdk1) to alanine revealed that this residue is required for viability. Subsequent studies demonstrated that when this Thr is mutated to Ala Cdk1 is inactive and, under some conditions, has cyclin-binding defects. Cdk1 is incapable of T161 autophosphorylation, but phosphorylation and kinase activation occur if Cdk1/cyclin complexes are incubated in crude cell extracts.

Purification of this CDK-activating kinase (CAK) activity from starfish oocytes revealed two major peptides that exist as a complex. Peptide microsequencing revealed that one of these proteins was highly homologous to the predicted product of the M015 gene cloned from a Xenopus cDNA library, but the other peptide could not be identified. Purified CAK, or M015 protein immunoprecipitated from extracts, was able to phosphorylate and activate Cdk1 and Cdk2 in vitro, and immunodepletion of M015 substantially decreased CAK activity in extracts. M015 was revealed to be a CDK itself; CAK purified from human HeLa cells contained two polypeptides in apparent 1:1 stoichiometry: 1) the human homolog of M015 and 2) a 37 kDa polypeptide homologous to cyclins. CAK activity could be reconstituted in vitro with recombinant M015 (renamed Cdk7) and the cyclin (cyclin H) purified from E. coli. Thus metazoan CAK is a complex of Cdk7 (M015) and cyclin H. Cdk7 homologs in Drosophila, C. elegans, and fission yeast are all CAKs and required for viability, but, surprisingly, the cell cycle defects due to impaired Cdk7 function were exclusively in activating Cdk1 and promoting mitotic entry. Therefore, it remained uncertain whether Cdk7 was the CDK activating kinase for both the early and late cell cycle transitions or if a second CAK exists in metazoan cells.

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