Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle

Abstract

Somara S, Gilmont RR, Varadarajan S, Bitar KN. Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle. Am J Physiol Gastrointest Liver Physiol 299: G1164 -G1176, 2010. First published September 9, 2010 doi:10.1152/ajpgi.00479.2009.-Small heat shock proteins HSP27 and HSP20 have been implicated in regulation of contraction and relaxation in smooth muscle. Activation of PKC-␣ promotes contraction by phosphorylation of HSP27 whereas activation of PKA promotes relaxation by phosphorylation of HSP20 in colonic smooth muscle cells (CSMC). We propose that the balance between the phosphorylation states of HSP27 and HSP20 represents a molecular signaling switch for contraction and relaxation. This molecular signaling switch acts downstream on a molecular mechanical switch [tropomyosin (TM)] regulating thin-filament dynamics. We have examined the role of phosphorylation state(s) of HSP20 on HSP27-mediated thin-filament regulation in CSMC. CSMC were transfected with different HSP20 phosphomutants. These transfections had no effect on the integrity of actin cytoskeleton. Cells transfected with 16D-HSP20 (phosphomimic) exhibited inhibition of acetylcholine (ACh)-induced contraction whereas cells transfected with 16A-HSP20 (nonphosphorylatable) had no effect on ACh-induced contraction. CSMC transfected with 16D-HSP20 cDNA showed significant decreases in 1) phosphorylation of HSP27 (ser78); 2) phosphorylation of PKC-␣ (ser657); 3) phosphorylation of TM and CaD (ser789); 4) ACh-induced phosphorylation of myosin light chain; 5) ACh-induced association of TM with HSP27; and 6) ACh-induced dissociation of TM from caldesmon (CaD). We thus propose the crucial physiological relevance of molecular signaling switch (phosphorylation state of HSP27 and HSP20), which dictates 1) the phosphorylation states of TM and CaD and 2) their dissociations from each other.relaxation; heat shock protein; molecular signaling switch; molecular mechanical switch; smooth muscle contraction AGONIST-INDUCED INCREASES in intracellular Ca 2ϩ concentrations have been implicated in the initiation of smooth muscle contraction (42,46, 54,61). Although phosphorylation of regulatory myosin light chains (MLC 20 ) by activation of Ca 2ϩ / calmodulin-dependent myosin light chain kinase (MLCK) is the key mechanism for initiation of smooth muscle contraction, inactivation of myosin light chain phosphatase (MLCP) is the key mechanism for maintenance of smooth muscle contraction (25). However, maintenance of force upon decrease in intracellular calcium concomitant with decrease in phosphorylation of MLC 20 is associated with thin-filament regulation (18,36,38,64). Thin-filament regulation explains the maintenance of tone at low MLC 20 phosphorylation. Thin-filament regulation modulates the interaction of actin molecules of the thin filament with myosin heads of the thick filament for contraction to occur (10,20,53,65).Relaxation of smooth muscle occurs either as a result of removal of the con...

Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC)

“…In a similar manner we were also able to corroborate, with our datasets, the phosphorylation of proteins also previously associated with muscarinic receptor signaling cascades, e.g . mitogen-activated protein kinase 1 [30], myristoylated alanine-rich C-kinase substrate [31] and heat-shock protein 27kDa [32]. Therefore our identified phosphorylation events appear to be consistent with expected results of experimental receptor stimulation.…”

Role of Cyclic Nucleotide-Dependent Actin Cytoskeletal Dynamics: [Ca2+]i and Force Suppression in Forskolin-Pretreated Porcine Coronary Arteries

“…Phosphorylated HSP20 has been shown to promote airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin [25], [51]. In colonic smooth muscle, HSP20 is activated by PKA and modulates the association of caldesmon and tropomyosin during the maintenance of tone [52]. HSP20 may act as the functional switch for contraction and relaxation in the colonic smooth muscle [52].…”

“…In colonic smooth muscle, HSP20 is activated by PKA and modulates the association of caldesmon and tropomyosin during the maintenance of tone [52]. HSP20 may act as the functional switch for contraction and relaxation in the colonic smooth muscle [52]. HSP20-mediated force suppression may indeed involve different mechanisms in smooth muscle from various types and species.…”

Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering

“…Rabbit colonic circular smooth muscle cells (RCSMCs) were isolated as described previously [23]. Briefly, rabbit sigmoid colon was cleaned of fecal content.…”