Identification of Substrate Binding Site of Cyclin-dependent Kinase 5*

Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis (“charged to alanine”) and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.

[1]  Zhou Songyang,et al.  Use of an oriented peptide library to determine the optimal substrates of protein kinases , 1994, Current Biology.

[2]  Mingjie Zhang,et al.  Cyclin-dependent Kinase 5 (Cdk5) Activation Domain of Neuronal Cdk5 Activator , 1997, The Journal of Biological Chemistry.

[3]  N. Metropolis,et al.  Equation of State Calculations by Fast Computing Machines , 1953, Resonance.

[4]  Veeranna,et al.  Mitogen-Activated Protein Kinases (Erk1,2) Phosphorylate Lys-Ser-Pro (KSP) Repeats in Neurofilament Proteins NF-H and NF-M , 1998, The Journal of Neuroscience.

[5]  P. Jeffrey,et al.  Structural basis of cyclin-dependent kinase activation by phosphorylation , 1996, Nature Structural Biology.

[6]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[7]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[8]  L. Johnson,et al.  Protein kinase inhibition by staurosporine revealed in details of the molecular interaction with CDK2 , 1997, Nature Structural Biology.

[9]  Alexander D. MacKerell,et al.  A molecular mechanics force field for NAD+ NADH, and the pyrophosphate groups of nucleotides , 1997, J. Comput. Chem..

[10]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[11]  R. Aebersold,et al.  A brain-specific activator of cyclin-dependent kinase 5 , 1994, Nature.

[12]  M. Levitt.  Accurate modeling of protein conformation by automatic segment matching. , 1992, Journal of molecular biology.

[13]  L. Tsai,et al.  p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5 , 1994, Nature.

[14]  D O Morgan,et al.  Cyclin-dependent kinases: engines, clocks, and microprocessors. , 1997, Annual review of cell and developmental biology.

[15]  L. Johnson,et al.  Active and Inactive Protein Kinases: Structural Basis for Regulation , 1996, Cell.

[16]  T. Hunt,et al.  The crystal structure of cyclin A. , 1995, Structure.

[17]  T. Hunter,et al.  Identification of Functional Domains in the Neuronal Cdk5 Activator Protein* , 1997, The Journal of Biological Chemistry.

[18]  T. Soderling,et al.  A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1 , 1996, Molecular and cellular biology.

[19]  Bernard R. Brooks,et al.  New spherical‐cutoff methods for long‐range forces in macromolecular simulation , 1994, J. Comput. Chem..

[20]  Sung-Hou Kim,et al.  Crystal structure of cyclin-dependent kinase 2 , 1993, Nature.

[21]  J. Zheng,et al.  Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[22]  H. Pant,et al.  cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. H. Wang,et al.  Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain. , 1993, The Journal of biological chemistry.

[24]  L. Johnson,et al.  The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition , 1997, The EMBO journal.

[25]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[26]  John A Tainer,et al.  Crystal Structure and Mutational Analysis of the Human CDK2 Kinase Complex with Cell Cycle–Regulatory Protein CksHs1 , 1996, Cell.

[27]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[28]  L. Johnson,et al.  The cyclin box fold: protein recognition in cell-cycle and transcription control. , 1997, Trends in biochemical sciences.

[29]  Kornelia Polyak,et al.  Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex , 1995, Nature.