Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro.
The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.
Manenti
S
S
INSERM CJF 95-10, IFR 30, Hôpital Purpan, Pavillon Rayer, 31059 Toulouse, France. manenti@purpan.inserm.fr
Yamauchi
E
E
Sorokine
O
O
Knibiehler
M
M
Van Dorsselaer
A
A
Taniguchi
H
H
Ducommun
B
B
Darbon
J M
JM
D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D016384:Consensus Sequence / Q000235:genetics; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D019927:Cyclin E; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D010769:Phosphothreonine; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase