Phosphorylation sequences in h‐caldesmon from phorbol ester‐stimulated canine aortas

Abstract

The high molecular weight form of caldesmon (h‐caldesmon) is phosphorylated in vascular smooth muscle. The stoichiometry of caldesmon phosphorylation increases in response to stimulation of the muscle by several contractile agonists; however, the responsible kinase has not been identified. In this study, we have sequenced the phosphopeptides prepared from h‐caldesmon phosphorylated in vitro by protein kinase C (PKC) as well as the phosphopeptides prepared from caldesmon phosphorylated in intact canine aortas that were stimulated to contract with PDBu. PKC phosphorylated three sites located in the C terminus: GSS*LKIEE, AEFLNKS*VQK and NLWEKQS*VDK, while h‐caldesmon from intact tissue was phosphorylated at two separate sites also in the C terminus: VTS*PTKV and S*PAPK. By comparison to known substrate consensus sequences for various protein kinases these data suggest that h‐caldesmon is directly phosphorylated by a proline‐directed protein kinase and not by PKC.

Mammal-specific, ERK-dependent, Caldesmon Phosphorylation in Smooth Muscle

“…l-CaD is present in most cells, where it is hypothesized to regulate the dynamics of actin filament organization (8); however, the possibility remains that the function of both l-CaD and h-CaD is similar and may involve the regulation of some process other than cell shortening. Because both isoforms of CaD are phosphorylated in cells (4,5,8,9,(13)(14)(15)(16), it is likely that the covalent addition and removal of phosphate serve to regulate its function in a reversible manner. A better understanding of the phosphorylation process should help to determine the physiological role of the protein in tissue.…”

“…Although the identities of all phosphorylation sites in h-CaD have not been elucidated, phosphopeptide sequencing of the protein isolated from 32 P-loaded muscle shows that phosphate is contained in Ser 759 and Ser 789 , based on the numbering of the human CaD sequence (6). Approximately two-thirds of the total amount of phosphate in h-CaD is incorporated into these two ERK-dependent sites, whereas the site(s) of the remaining one-third are unknown (5). Using the newly developed phosphopeptide-specific antibodies designed to monitor the phosphorylation levels in Ser 759 and Ser 789 , the major site of ERK-dependent phosphorylation in h-CaD was determined to be at Ser…”

“…In an attempt to identify a switch that may regulate caldesmon function in tissue, we previously showed that the protein is phosphorylated in resting smooth muscle and that the level of phosphorylation increases with muscle stimulation by many chemical agents, including phorbol esters, endothelin-1, histamine, and okadaic acid, and by depolarization with KCl (4). Sequencing of phosphopeptides generated from h-CaD purified from 32 P-labeled arterial smooth muscle led to the identification of at least two sites of phosphorylation, Ser 759 and Ser 789 (5), based on the numbering of the mammalian sequence (6), both in the carboxyl terminus of the protein. The site at Ser 759 is conserved in gizzard h-CaD (equivalent to Ser 702 ); however, the site at Ser 789 is absent from the gizzard protein.…”

“…Previously reported results from sequencing experiments showed that both Ser 759 and Ser 789 of h-CaD contained phosphate when the protein was extracted from arterial muscle (5). These experiments did not seek to determine the amount of phosphate incorporated into each of the two sites and at which site, if either, there was a modulation of phosphate content during muscle stimulation.…”

Ras proteins increase Ca2+‐responsiveness of smooth muscle contraction

“…which is associated with an increase in myosin light chain phosphorylation and inhibition of phosphatase [7]. MAP2 kinase phosphorylates caldesmon [29,30], which results in weakening of the binding of caldesmon to actin [29]. Assuming that unphosphorylated caldesmon inhibits smooth muscle contraction [3 1.321, phosphorylation of caldesmon may be a mechanism to increase Ca'+-sensitivity independent of an increase in myosin light chain phosphorylation [33].…”