Role of the pyrrolidine ring of proline in determining the substrate specificity of cdc2 kinase or cdk5.
To examine structural features of proline which are essential for the proline-directed phosphorylation by cdc2 kinase or cdk5, we prepared the peptide representing the cdc2 kinase phosphorylation site at Ser-55 in vimentin [Ser-Leu-Tyr-Ser-Ser-Ser55-Pro56-Gly-Gly58-Ala-Tyr-NH2], the peptide containing arginine in place of Gly-58, and their derivatives containing various N-methylamino acids or proline homologs in place of Pro-56, and tested them as substrates for the kinases. While substitution of the proline by proline homologs (L-pipecolic acid or L-azetidine-2-carboxylic acid) increased the K(m) value 2- to 4-fold at utmost, substitution by N-methylamino acids (sarcosine, L-N-methylalanine, L-N-methylvaline, or L-N-methylleucine) increased the K(m) value 7- to 40-fold for cdc2 kinase. For cdk5, these substitutions led to parallel effects on the K(m) value to those found for cdc2 kinase; cdk5 recognized the peptides with a proline specificity similar to that for cdc2 kinase. These results suggest that the pyrrolidine ring of proline is important for substrate recognition by cdc2 kinase or cdk5. Molecular dynamics and molecular mechanics simulations indicated that the pyrrolidine ring of proline is optimal to stabilize a beta-turn at the phosphorylation site and that the K(m) values of the peptides for the enzymes might be related to the probability of the turn structure. The results obtained here also suggest that the pyrrolidine ring of proline is required to maintain a high V(max) value for cdc2 kinase or especially for cdk5. These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.
Ando
S
S
Laboratory of Chemistry, Saga Medical School. andoh-s@smsnet.saga-med.ac.jp
Ikuhara
T
T
Kamata
T
T
Sasaki
Y
Y
Hisanaga
S
S
Kishimoto
T
T
Ito
H
H
Inagaki
M
M
D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D003198:Computer Simulation; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011759:Pyrrolidines / Q000737:chemistry*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D014746:Vimentin / Q000378:metabolism
D009842:Oligopeptides; D011759:Pyrrolidines; D014746:Vimentin; D012694:Serine; D011392:Proline; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases