Mark J. Solomon

CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15.

The mitotic inducer p34cdc2 requires association with a cyclin and phosphorylation on Thr161 for its activity as a protein kinase. CAK, the p34cdc2 activating kinase, was previously identified as an enzyme necessary for this activating phosphorylation. We confirm here that CAK is a protein kinase and describe its purification over 13,000‐fold from Xenopus egg extracts. We further show that CAK contains a protein identical or closely related to the previously identified Xenopus MO15 gene: p40MO15 copurifies with CAK, and an antiserum to p40MO15 specifically depletes cAK activity. CAK appears to be the only protein in Xenopus egg extracts that can activate complexes of either p34cdc2 or the closely related protein kinase, p33cdk2, with either cyclin A or cyclin B. The sequence similarity between p40MO15 and p34cdc2, and the approximately 200 kDa size of CAK, suggest that p40MO15 may itself be regulated by subunit association and by protein phosphorylations.

The Cdk-Activating Kinase (CAK) from Budding Yeast

Activation of the cyclin-dependent kinases to promote cell cycle progression requires their association with cyclins as well as phosphorylation of a threonine (residue 161 in human p34cdc2). This phosphorylation is carried out by CAK, the Cdk-activating kinase. We have purified and cloned CAK from S. cerevisiae. Unlike CAKs from other organisms, Cak1p is active as a monomer, has full activity when expressed in E. coli, and is not a component of the basal transcription factor, TFIIH. A temperature-sensitive mutation in CAK1 confers a G2 delay accompanied by low Cdc28p protein kinase activity and shows genetic interactions with altered expression of the gene for the major mitotic cyclin, CLB2. Our data raise the intriguing possibility that p40MO15-cyclin H-MAT1, identified as the predominant CAK in vertebrate cell extracts, may not function as a physiological CAK.

Activation of the various cyclin/cdc2 protein kinases

Recent research has led to the near culmination of the biochemical confirmation of the most basic genetic predictions about how p34cdc2 activity is controlled. The field is now moving from a biochemical dissection of the machinery to understanding how the system is regulated. The discovery of numerous, highly related protein kinase complexes that control other cell cycle events will test the generality of this paradigm.

A Predictive Scale for Evaluating Cyclin-dependent Kinase Substrates A COMPARISON OF p34cdc2 AND p33cdk2

Protein phosphorylation by members of the Cdk (cyclin-dependent kinase) family of protein kinases is necessary for progression through the cell cycle. However, the primary sequence determinants of Cdk substrate specificity have yet to be examined quantitatively. We have used a panel of glutathione S-transferase peptide fusions to investigate the fine-structure specificity of p33cdk2 and p34cdc2. Our data indicate that the generally held consensus sequences for p34cdc2 represent a significant oversimplification of its true specificity and that this specificity is conserved between species. p33cdk2 and p34cdc2 have similar but distinct substrate specificities that are affected modestly by the associated cyclin subunit. We derive specific values of phosphorylation efficiencies by these enzymes that can be used to estimate the phosphorylation potential of proposed Cdk substrates.

Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes

Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control. We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of linker histones into nucleosomal array substrates. Much of this inhibition is independent of linker histone-induced folding of the arrays. We also find that phosphorylation of the linker histone by Cdc2/Cyclin B kinase can rescue remodeling by ySWI/SNF. These results suggest that linker histones exert a global, genome-wide control over remodeling activities, implicating a new, obligatory coupling between linker histone kinases and ATP-dependent remodeling enzymes.

Dephosphorylation of Human Cyclin-dependent Kinases by Protein Phosphatase Type 2Cα and β2 Isoforms

We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2Cβ2, a novel PP2Cβ isoform, and to PP2Cα. PP2Cα and PP2Cβ2 co-purified with Mg2+-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2Cα and PP2Cβ2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2Cα and PP2Cβ2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs.

Hsl1p, a Swe1p Inhibitor, Is Degraded via the Anaphase-Promoting Complex

ABSTRACT Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G1, accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations inCDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G1-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1pdb-mut) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCFMet30 in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.

Regulation of CDKs by phosphorylation

The key transitions of the cell cycle are controlled via the sequential activation and inactivation of members of the cyclin-dependent kinase (CDK) subfamily of protein kinases. The activities of these enzymes are regulated by multiple mechanisms including both activating and inactivating phosphorylations, binding to regulatory subunits termed cyclins, subcellular localization, and association with inhibitory proteins (CKIs). There are two broad classes of regulatory inputs. Regulation in response to intrinsic signals ensures the proper timing of the basic cell cycle and coordinates various cell cycle events via checkpoints that monitor the completion of each step. This regulation is responsible for the abrupt transitions between cell cycle phases as well as for the fidelity of the cell division process. Phosphorylation of CDKs is very important for such intrinsic regulation, although other mechanisms participate as well. In contrast, extrinsic signals impinge on the cell, resulting in stimulation or inhibition of the cell cycle. These signals typically affect the levels of CDK inhibitors.

The function(s) of CAK, the p34cdc2-activating kinase

The protein kinase p34cdc2 plays a central role in controlling the G2 to mitosis transition in all eukaryotic cells. It is regulated by protein-protein association and by multiple phosphorylations; one of these phosphorylations is absolutely required for activity. Until recently, the molecular identity of the protein kinase that phosphorylates this site was unknown. The subunits of this enzyme have been identified recently as p40MO15, the catalytic subunit, and cyclin H, a regulatory subunit. Similarities between this kinase and the p34cdc2 family of protein kinases suggest how p40MO15 itself may be regulated.

Identification and comparative analysis of multiple mammalian Speedy/Ringo proteins

In addition to their activation via binding to cyclins, cyclin-dependent kinases (CDKs)can be activated via binding to a novel cell cycle regulator termed Speedy or Ringo, whichshows no apparent similarity to cyclins. The first Speedy/Ringo protein was found to beessential for Xenopus oocyte maturation and a human homolog (Spy1, herein calledSpeedy/Ringo A1) regulates S-phase entry and cell survival after DNA damage in culturedsomatic cells. We have identified a Speedy/Ringo-like gene in the most primitive branchingclade of chordates (Ciona intestinalis), as well as four mammalian homologs. Of the mammalianproteins, two, Speedy/Ringo A and C, bind to Cdc2 and Cdk2, whereas Speedy/Ringo B bindspreferentially to Cdc2. Despite their distinct CDK-binding preferences, both Speedy/Ringo Aand B can promote the maturation of Xenopus oocytes and all three Speedy/Ringo proteins canbind to and activate CDKs in vivo. These mammalian Speedy/Ringo proteins exhibit distincttissue expression patterns, though all three are enriched in testis, consistent with the initialobservation that Xenopus Speedy/Ringo functions during meiosis. Speedy/Ringo A is widelyexpressed in tissues and cell lines. Speedy/Ringo B expression appears to be testis-specific.Speedy/Ringo C is expressed in diverse tissues, particularly those that undergo polyploidization.All Speedy/Ringo proteins share a highly conserved ~140-aa domain we term the Speedy/Ringobox that is essential for CDK binding. Point mutations in this domain abolish CDK binding.Besides the central Speedy/Ringo box, Speedy/Ringo A contains a C-terminal portion, whichpromotes CDK activation, and an N-terminal portion, which is dispersible for both CDK bindingand activation but that influences protein expression. The existence of this growing family ofCDK activators suggests that Speedy/Ringo proteins may play as complex a role in cell cyclecontrol as the diverse family of cyclins.