Phosphorylation of Caldesmon Prevents Its Interaction with Smooth Muscle Myosin

Abstract

Caldesmon is known to bind to smooth muscle myosin. Ca2+/calmodulin-dependent phosphorylation of caldesmon completely blocks its interaction with myosin. Cleavage of caldesmon at its 2 cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) occurs initially at one site to yield 108-kDa and 21.2-kDa peptides and subsequently at the second site within the 108-kDa peptide to yield 85-kDa and 23.5-kDa fragments. The 23.5-kDa peptide retains the ability to bind to myosin. The N-terminal (95 kDa) and C-terminal (42 kDa) chymotryptic peptides of caldesmon were isolated and digested with NTCB: the C-terminal actin- and calmodulin-binding peptide was not cleaved, indicating that it does not contain either of the cysteine residues, whereas the 95-kDa N-terminal peptide was cleaved at two sites to yield 56-kDa, 23.5-kDa, and 21.2-kDa fragments. The arrangement of NTCB fragments in caldesmon is, therefore: 21.2 kDa/23.5 kDa/85 kDa from N to C terminus. Digestion of phosphorylated caldesmon with NTCB suggested a single phosphorylation site in the 21.2-kDa peptide and three sites in the 23.5-kDa peptide. These results lead to the development of a model whereby caldesmon may cross-link actin to myosin and such cross-linking is blocked by phosphorylation of caldesmon. This mechanism may explain the formation of reversible "latch bridges" which permit force maintenance at low levels of myosin phosphorylation in intact smooth muscles.