Phosphorylation of troponin I by protein kinase C: mechanism of inhibition by calmodulin and troponin C.
The mechanism by which calmodulin and troponin C influence phosphorylation of troponin I (TnI) by protein kinase C was investigated. The phosphorylation of TnI by protein kinase C requires the presence of acidic phospholipid, calcium and diacylglycerol. Light scattering intensity and fluorescence intensity experiments showed that TnI associated with the phospholipid membranes and caused extensive aggregation. In the presence of Ca2+, TnI-phospholipid interactions were prevented by approximately stoichiometric amounts of either troponin C or calmodulin. Troponin C was shown to completely inhibit phosphorylation of TnI by either protein kinase C or by phosphorylase b kinase. In contrast, calmodulin completely inhibited phosphorylation of TnI by protein kinase C, but had only little effect on TnI phosphorylation by phosphorylase b kinase. Inhibition by calmodulin did not appear to be due to interaction with PKC, since calmodulin mildly increased protein kinase C phosphorylation of histone III-S. The ratio of phosphoserine to phosphothreonine in protein kinase C-phosphorylated TnI remained approximately constant for reactions inhibited by up to 90% by calmodulin. TnI interactions with phospholipid and phosphorylation of TnI by PKC were also prevented by high salt concentrations. However, salt concentrations adequate to inhibit phosphorylation were sufficient to dissociate only TnI, but not protein kinase C from the membrane. These results suggest that the binding of TnI to phospholipid is required for phosphorylation by protein kinase C and that prevention of this binding by any means completely inhibited phosphorylation of TnI by protein kinase C.
Bazzi
M D
MD
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Lampe
P D
PD
Strasburg
G M
GM
Nelsestuen
G L
GL
D000818:Animals; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D008081:Liposomes / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014336:Troponin / Q000378:metabolism* / Q000494:pharmacology*; D019209:Troponin C; D019210:Troponin I
D002147:Calmodulin; D008081:Liposomes; D010743:Phospholipids; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D012965:Sodium Chloride; D011493:Protein Kinase C