The Actin Binding Protein, Fesselin, is a Member of the Synaptopodin Family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins. Originally published Biochem Biophys Res Commun. Vol. 371, No. 3, July 200

Phosphorylation of Caldesmon at Sites between Residues 627 and 642 Attenuates Inhibitory Activity and Contributes to a Reduction in Ca2+-Calmodulin Affinity

Caldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca2+-calmodulin. We investigated the effects of phosphorylation by p(21)-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca2+-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca2+-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca2+-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca2+-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity

Augmented contractility of murine femoral arteries in a streptozotocin diabetes model is related to increased phosphorylation of MYPT1

Diabetes mellitus (DM) is a metabolic disorder with high prevalence, and a major risk factor for macro- and microvascular abnormalities. This study was undertaken to explore the mechanisms of hypercontractility of murine femoral arteries (FA) obtained from mice with streptozotocin (STZ)-induced diabetes and its relation to the phosphorylation profile of the myosin phosphatase target subunit 1, MYPT1. The immunoreactivity of MYPT1 toward phospho-MYPT1-T696, MYPT1-T853, or MYPT1-S695, used as a read out for MYPT1 phosphorylation, has been studied by Western Blotting. Contractile activity of FA from control and STZ mice has been studied by wire myography. At basal conditions (no treatment), the immunoreactivity of MYPT1-T696/T853 was similar to 2-fold higher in the STZ arteries compared with controls. No changes in MYPT1-T696/853 phosphorylation were observed after stimulation with the Thromboxan-A(2) analog, U46619. Neither basal nor U46619-stimulated phosphorylation of MYPT1 at S695 was affected by STZ treatment. Mechanical distensibility and basal tone of FA obtained from STZ animals were similar to controls. Maximal force after treatment of FA with the contractile agonists phenylephrine (10 mu mol/L) or U46619 (1 mu mol/L) was augmented in the arteries of STZ mice by similar to 2- and similar to 1.5-fold, respectively. In summary, our study suggests that development of a hypercontractile phenotype in murine FA in STZ diabetes is at least partially related to an increase in phosphorylation of MLCP at MYPT1-T696/853. Interestingly, the phosphorylation at S695 site was not altered in STZ-induced diabetes, supporting the view that S695 may serve as a sensor for mechanical activity which is not directly involved in tone regulation

The Actin Binding Protein Fesselin is a Member of the Synaptopodin Family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively nfolded proteins fesselin has multiple binding partners including actin myosin calmodulin and ŽÂ±-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations uggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have etermined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman nalysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human ynaptopodin 2 and belongs to the synaptopodin family of proteins. Originally published Biochem Biophys Res Commun. Vol. 371 No. 3 July 200