A Mutation in the Human Cyclin-dependent Kinase Interacting Protein, CksHs2, Interferes With Cyclin-dependent Kinase Binding and Biological Function, but Preserves Protein Structure and Assembly (original) (raw)
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Interactions of Cyclins with Cyclin-Dependent Kinases: A Common Interactive Mechanism
Biochemistry, 1997
The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 form highly stable complexes with their "natural" cyclin partners with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and cdk7-cyclin A complexes present a 25-fold lower stability. On the basis of both the structure of the cdk2-cyclin A complex and on our kinetic results, we suggest that interaction of any cyclin with any cdk involves the same hydrophobic contacts and induces a marked conformational change in the catalytic cleft of the cdks. Although cdks bind ATP strongly, they remain in a catalytically inactive conformation. In contrast, binding of the cyclin induces structural rearrangements which result in the selective reorientation of ATP, a concomitant 3-fold increase in its affinity, and a 5-fold decrease of its release from the active site of cdks. . ‡ These authors contributed equally to this work. X Abstract published in AdVance ACS Abstracts, April 1, 1997. 1 Abbreviations: cdk, cyclin-dependent kinase; CAK, cdks activating kinase; MAPK, mitogen-activated protein kinase; cPKA, cyclic-AMPdependent protein kinase; CK1, casein kinase 1; mant-ATP and mant-ADP, N-methylanthraniloyl-adenosine di and triphosphate; GST, glutathione S-transferase.
Recent developments in cyclin-dependent kinase biochemical and structural studies
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2010
The cyclin-dependent kinases (CDKs) have been intensely studied because of their involvement in regulating essential cellular activities that include proliferation and transcription. A series of CDK2-containing structures have informed a general model for the molecular details of CDK activation and regulation. Recent structural studies of other members of the CDK family have lead to a re-appraisal of this model. In this review, we describe alternative CDK-cyclin assemblies taking the recently characterised CDK/cyclin complexes, CDK9/cyclinT1 and CDK4/cyclinD as examples. The differential effects of CDK phosphorylation on CDK activation state and substrate specificity are examined in the light of recent data on CDK2/cyclinA, CDK9/cyclinT, CDK4/cyclinD and Pho85/Pho80. We also present an overview of factors that affect CDK substrate specificity, and, in particular, the contributions that are made by the cyclin subunit. Finally, we review recent results that have helped to unravel the molecular mechanisms underlying the conflicting roles of the Cip/Kip CDK inhibitor family in CDK regulation.
Cyclin-dependent kinases: inhibition and substrate recognition
Current Opinion in Structural Biology, 1999
Four unresolved issues of cyclin-dependent kinase (CDK) regulation have been addressed by structural studies this year -the mechanism of CDK inhibition by members of the INK4 family of CDK inhibitors, consensus substrate sequence recognition by CDKs, the role of the cyclin subunit in substrate recognition and the structural mechanism underlying CDK inhibition by phosphorylation.
Biochemical Characterization of the Human Cyclin-dependent Protein Kinase Activating Kinase
Journal of Biological Chemistry, 1996
The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.
The Cyclin-dependent Kinases cdk2 and cdk5 Act by a Random, Anticooperative Kinetic Mechanism
Journal of Biological Chemistry, 2001
cdk2⅐cyclin E and cdk5⅐p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2⅐GST-cyclin E and cdk5⅐GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K i value of 0.6 mM. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K i value of 2 M. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2⅐GST-cyclin E the kinetic parameters were determined to be K m, ATP ؍ 3.6 ؎ 1.0 M, K m, peptide ؍ 4.6 ؎ 1.4 M, and the anticooperativity factor, ␣ ؍ 130 ؎ 44. For cdk5⅐GST-p25, the K m, ATP ؍ 3.2 ؎ 0.7 M, K m, peptide ؍ 1.6 ؎ 0.3 M, and ␣ ؍ 7.2 ؎ 1.8. Kinases are a major component of the signal transduction pathways involved in cellular regulation. In addition to their role in maintaining normal homeostasis, there is increasing evidence implicating these enzymes in various diseases, such * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 1JSV) have been deposited in the Protein Data Bank,
1997
Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (a and/or a') and two non-catalytic, P-subunits. The B-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191 -198 regions of the a-subunit, the negative regulation by the P-subunit and by its N-terminal synthetic fragment CK2P-( 1 -77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal, CK2B-(155 -215)-peptide is unaffected or even increased. Moreover, the basal activity of (x mutants K74-77A, K79R80K83A, and R191R195K198A toward specific peptide substrates is stimulated by the P-subunit many fold more than that of a wild type, while extrastimulation by p mutant DSSLShE57A, observable with a wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of P is mediated by basic residues in the 74-83 and in the 191 -198 sequences of the a-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the p subunit operates as a pseudosubstrate. In contrast, another CK2rx mutant, V66A, is more sensitive to inhibition by either P-subunit or its N-terminal, CK2P-(1 -77)-peptide, while its stiinulation by the C-terminal peptide, CK2P-(ISS-21 S), is comparable to that of rx wild type. These observations suggest an indirect role of Val66 in conferring to the a-subunit a conformation less sensitive to down regulation by B-subunit.
Selectivity and potency of cyclin-dependent kinase inhibitors
The AAPS Journal, 2006
Members of the cyclin-dependent kinase (CDK) family play key roles in various cellular processes. There are 11 members of the CDK family known till now. CDKs are activated by forming noncovalent complexes with cyclins such as A-, B-, C-, D-(D1, D2, and D3), and E-type cyclins. Each isozyme of this family is responsible for particular aspects (cell signaling, transcription, etc) of the cell cycle, and some of the CDK isozymes are specifi c to certain kinds of tissues. Aberrant expression and overexpression of these kinases are evidenced in many disease conditions. Inhibition of isozymes of CDKs specifi cally can yield benefi ciary treatment modalities with minimum side effects. More than 80 3-dimensional structures of CDK2, CDK5, and CDK6 complexed with inhibitors have been published. This review provides an understanding of the structural aspects of CDK isozymes and binding modes of various known CDK inhibitors so that these kinases can be better targeted for drug discovery and design. The amino acid residues that constitute the cyclin binding region, the substrate binding region, and the area around the adenosine triphosphate (ATP) binding site have been compared for CDK isozymes. Those amino acids at the ATP binding site that could be used to improve the potency and subtype specifi city have been described.