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 e xamine this process, we bacterially expressed wild type TnI and TnI mu tants in which serine 22 and 23, a putative protein kinase A (PKA) sit e, and threonine 143, a putative protein kinase C (PKC) site, were rep laced by alanine S22A/23A and T143A. PKA dependent phosphorylation was similar to 90% reduced in the S22A/23A mutant and unaffected in T143A . PKC dependent phosphorylation was markedly reduced in T143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calci um sensitivity (i.e. inhibition of actomyosin ATPase in the presence o f EGTA) and regulation of the reconstituted actomyosin system was pres erved 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 calci um dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and thi s was markedly attenuated when T143 A was reassociated (although a sli ght rightward shift and a reduction in maximal ATPase activity was sti ll 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 respons e although other residues may also have functional significance.