A phosphorylation-independent role for the yeast cyclin-dependent kinase activating kinase Cak1

Cdc28 is the main cyclin-dependent kinase (CDK) directing the cell cycle in the budding yeast Saccharomyces cerevisiae. Besides cyclin binding, Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We have previously isolated carboxy-terminal cdc28^CST mutants that are temperature sensitive and exhibit high chromosome instability. Both phenotypes are suppressed by high copy Cak1 in a manner that is independent of its catalytic activity and conversely, combination of cdc28^CST and cak1 mutations results in synthetic lethality. Altogether, these results suggest that for the Cdc28 complexes to remain stable and active, an interaction with Cak1 is needed via the carboxyl terminus of Cdc28. We report two-hybrid assay data that support this model, and results that indicate that actively growing yeast cells require an optimum Cdc28:Cak1 ratio. While Cak1 is constitutively active and expressed, dividing cells tightly regulate Cak1 protein levels to ensure presence of adequate levels of Cdc28 CDK activity.     

Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.     

Cdc37 engages in stable,S14A mutation-reinforced association with the most atypical member of the yeast kinome,Cdk-activating kinase (Cak1)

In most eukaryotes, Cdc37 is an essential chaperone, transiently associating with newly synthesised protein kinases in order to promote their stabilisation and activation. To determine whether the yeast Cdc37 participates in any stable protein interactions in vivo, genomic two-hybrid screens were conducted using baits that are functional as they preserve the integrity of the conserved N-terminal region of Cdc37, namely a Cdc37-Gal4 DNA binding domain (BD) fusion in both its wild type and its S14 nonphosphorylatable (Cdc37(S14A)) mutant forms. While this failed to identify the protein kinases previously identified as Cdc37 interactors in pull-down experiments, it did reveal Cdc37 engaging in a stable association with the most atypical member of the yeast kinome, cyclin-dependent kinase (Cdk1)-activating kinase (Cak1). Phosphorylation of the conserved S14 of Cdc37 is normally crucial for the interaction with, and stabilisation of, those protein kinase targets of Cdc37, Cak1 is unusual in that the lack of this Cdc37 S14 phosphorylation both reinforces Cak1:Cdc37 interaction and does not compromise Cak1 expression in vivo. Thus, this is the first Cdc37 client kinase found to be excluded from S14 phosphorylation-dependent interaction. The unusual stability of this Cak1:Cdc37 association may partly reflect unique structural features of the fungal Cak1.     

Activation of a nuclear Cdc2-related kinase within a mitogen-activated protein kinase-like TDY motif by autophosphorylation and cyclin-dependent protein kinase-activating kinase

Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.     

Fission yeast Csk1 is a CAK-activating kinase (CAKAK).

Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).     

The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.     

Chemical clamping allows for efficient phosphorylation of the RNA carrier protein Npl3

Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low Km values) through stabilization of the enzyme-substrate complex, these contacts could reduce catalytic turnover (decrease kcat) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a Km that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low Km through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.     

Phosphorylation of Ime2 regulates meiotic progression in Saccharomyces cerevisiae

Ime2p is a meiosis-specific protein kinase in Saccharomyces cerevisiae that controls multiple steps in meiosis. Although Ime2p is functionally related to the Cdc28p cyclin-dependent kinase (CDK), no cyclin binding partners that regulate its activities have been identified. The sequence of the Ime2p catalytic domain is similar to CDKs and mitogen-activated protein kinases (MAPKs). Ime2p is activated by phosphorylation of its activation loop in a Cak1p-dependent fashion and is subsequently phosphorylated on multiple residues as cells progress through meiosis. In this study, we show that Ime2p purified from meiotic cells is phosphorylated on Thr(242) and Tyr(244) in its activation loop and on Ser(520) and Ser(625) in its C terminus. Ime2p autophosphorylates on threonine in its activation loop in vitro consistent with autophosphorylation of Thr(242) playing a role in its activation. Moreover, autophosphorylation in cis is required for Ime2p to become hyperphosphorylated. Phosphorylation of the C-terminal serines is not essential to sporulation. However, Ime2p C-terminal phosphorylation site mutants genetically interact with components of the FEAR network that controls exit from meiosis I. These data suggest that Ime2p plays a role in controlling the exit from meiosis I and demonstrate that a phospho-modification pathway regulates Ime2p during the different phases of meiotic development.     

Kinetic mechanism of fully activated S6K1 protein kinase

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His(6)-S6K1alphaII(DeltaAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (K(d)(ATP)=5-6 microM) was followed by low affinity binding of Tide (K(d)(Tide)=180 microM), and values of K(m)(ATP)=5-6 microM and K(m)(Tide)=4-5 microM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.     

The Cdk-activating kinase Cak1p promotes meiotic S phase through Ime2p

CAK1 encodes an essential protein kinase in Saccharomyces cerevisiae that is required for activation of the Cdc28p Cdk. CAK1 also has several CDC28-independent functions that are unique to meiosis. The earliest of these functions is to induce S phase, which is regulated differently in meiosis than in mitosis. In mitosis, Cdc28p controls its own S-phase-promoting activity by signaling the destruction of its inhibitor, Sic1p. In meiosis, Sic1p destruction is signaled by the meiosis-specific Ime2p protein kinase. Our data show that Cak1p is required to activate Ime2p through a mechanism that requires threonine 242 and tyrosine 244 in Ime2p's activation loop. This activation promotes autophosphorylation and accumulation of multiply phosphorylated forms of Ime2p during meiotic development. Consistent with Cak1p's role in activating Ime2p, cells lacking Cak1p are deficient in degrading Sic1p. Deletion of SIC1 or overexpression of IME2 can partially suppress the S-phase defect in cak1 mutant cells, suggesting that Ime2p is a key target of Cak1p regulation. These data show that Cak1p is required for the destruction of Sic1p in meiosis, as in mitosis, but in meiosis, it functions through a sporulation-specific kinase.     

Autophosphorylation of the Smk1 MAPK is spatially and temporally regulated by Ssp2 during meiotic development in yeast

Smk1 is a meiosis-specific MAPK that controls spore wall morphogenesis in Saccharomyces cerevisiae. Although Smk1 is activated by phosphorylation of the threonine (T) and tyrosine (Y) in its activation loop, it is not phosphorylated by a dual-specificity MAPK kinase. Instead, the T is phosphorylated by the cyclin-dependent kinase (CDK)–activating kinase, Cak1. The Y is autophosphorylated in an intramolecular reaction that requires a meiosis-specific protein named Ssp2. The meiosis-specific CDK-like kinase, Ime2, was previously shown to positively regulate Smk1. Here we show that Ime2 activity is required to induce the translation of SSP2 mRNA at anaphase II. Ssp2 protein is then localized to the prospore membrane, the structure where spore wall assembly takes place. Next the carboxy-terminal portion of Ssp2 forms a complex with Smk1 and stimulates the autophosphorylation of its activation-loop Y residue. These findings link Ime2 to Smk1 activation through Ssp2 and define a developmentally regulated mechanism for activating MAPK at specific locations in the cell.     

Transient-state kinetic analysis of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase reveals that a step after chemical transformation is rate limiting

MyristoylCoA:protein N-myristoyltransferase is a member of the superfamily of GCN5-related N-acetyltransferases and catalyzes the covalent attachment of myristate to the N-terminal Gly residue of proteins with diverse functions. Saccharomyces cerevisiae Nmt1p is a monomeric protein with an ordered bi-bi reaction mechanism: myristoylCoA is bound prior to peptide substrate; after catalysis, CoA is released followed by myristoylpeptide. Analysis of the X-ray structure of Nmt1p with bound substrate analogues indicates that the active site contains an oxyanion hole and a catalytic base and that catalysis proceeds through the nucleophilic addition-elimination mechanism. To determine the rate-limiting step in the enzyme reaction, pre-steady-state kinetic analyses were performed using a new, sensitive nonradioactive assay that detects CoA. Multiple turnover quenched flow studies disclosed that a step after the chemical transformation limits the overall rate of the reaction. Multiple and single turnover analyses revealed that the rate for the chemical transformation step is 13.8+/-0.6 s(-1) while the slower steady-state phase is 0.10+/-0.01 s(-1). Stopped flow kinetic studies of substrate acquisition indicated that binding of myristoylCoA to the apo-enzyme occurs through at least a two-step process, with a fast phase rate of 3.2 x 10(8) M(-1) s(-1) and a slow phase rate of 23+/-2 s(-1) (defined at 5 degrees C). Binding of an octapeptide substrate, representing the N-terminal sequence of a known yeast N-myristoylprotein (Cnb1p), to a binary complex composed of Nmt1p and a nonhydrolyzable myristoylCoA analogue (S-(2-oxo)pentadecylCoA) has a second-order rate constant of 2.1+/-0.3 x 10(6) M(-1) s(-1) and a dissociation rate of 26+/-15 s(-1) (defined at 10 degrees C). These results are interpreted in light of the X-ray structures of this enzyme.