Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) by proline-directed protein kinases and its dephosphorylation.
We identified two major substrates for the proline-directed protein kinases--cdc2 kinase and tau protein kinase II (TPKII)--in the cytosol fraction from rat brains. The molecular masses of the proteins were 80 and 46 kDa. Because the 80-kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine-rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80-kDa protein and purified MARCKS, we concluded that the 80-kDa protein is indeed MARCKS. The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two-dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline-directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it. These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.
Yamamoto
H
H
Department of Pharmacology, Kumamoto University School of Medicine, Japan.
Arakane
F
F
Ono
T
T
Tashima
K
K
Okumura
E
E
Yamada
K
K
Hisanaga
S
S
Fukunaga
K
K
Kishimoto
T
T
Miyamoto
E
E
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D051360:Cyclin-Dependent Kinase 5; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D038461:Proline-Directed Protein Kinases; D011493:Protein Kinase C / Q000378:metabolism; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats
D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D038461:Proline-Directed Protein Kinases; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; C079134:tau protein kinase II; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2