Expression and regulation of mutant forms of cardiac TnI in a reconstituted actomyosin system: role of kinase dependent phosphorylation.
When phosphorylated, the inhibitory subunit of troponin (TnI) causes a loss in calcium sensitivity and a decrease in actomyosin ATPase. To examine this process, we bacterially expressed wild type TnI and TnI mutants in which serine 22 and 23, a putative protein kinase A (PKA) site, and threonine 143, a putative protein kinase C (PKC) site, were replaced by alanine S22A/23A and TI43A. PKA dependent phosphorylation was approximately 90% reduced in the S22A/23A mutant and unaffected in T143A. PKC dependent phosphorylation was markedly reduced in T 143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calcium sensitivity (i.e. inhibition of actomyosin ATPase in the presence of EGTA) and regulation of the reconstituted actomyosin system was preserved in the absence of phosphorylation using wild type TnI or either mutant. Calcium sensitivity was decreased by both PKA and PKC with the wild type TnI but was unaffected by PKA when the S22A/23A mutant was employed and by PKC when the T143A mutant was reconstituted. The calcium dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and this was markedly attenuated when T143 A was reassociated (although a slight rightward shift and a reduction in maximal ATPase activity was still seen). These data confirm that phosphorylation of TnI by regulatory kinases plays a major role in the regulation of myofibrillar ATPase. The N-terminal serines (22 and 23) appear to be uniquely important for the PKA response whereas threonine 143 is involved in the PKC response although other residues may also have functional significance.
Malhotra
A
A
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Nakouzi
A
A
Bowman
J
J
Buttrick
P
P
D000205:Actomyosin / Q000302:isolation & purification / Q000378:metabolism*; D000409:Alanine; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013912:Threonine; D019210:Troponin I / Q000096:biosynthesis / Q000378:metabolism*
D011994:Recombinant Proteins; D019210:Troponin I; D013912:Threonine; D000205:Actomyosin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D009218:Myosins; D000409:Alanine; D002118:Calcium