Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C.
Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.
Sahin
Bogachan
B
Department of Psychiatry, Protein Chemistry Laboratory, Alliance for Cellular Signaling, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Shu
Hongjun
H
Fernandez
Joseph
J
El-Armouche
Ali
A
Molkentin
Jeffery D
JD
Nairn
Angus C
AC
Bibb
James A
JA
D000818:Animals; D001921:Brain / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016716:PC12 Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D012694:Serine; D013379:Substrate Specificity
D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C021042:protein phosphatase inhibitor-1; C472592:protein phosphatase inhibitor-1, rat; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C