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Phosphorylation of caldesmon at sites between residues 627 and 642 attenuates inhibitory activity and contributes to a reduction in Ca2+-calmodulin affinity.

ABSTRACT: 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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca(2+)-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca(2+)-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.

SUBMITTER: Hamden SS

PROVIDER: S-EPMC2940999 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Phosphorylation of caldesmon at sites between residues 627 and 642 attenuates inhibitory activity and contributes to a reduction in Ca2+-calmodulin affinity.

Hamden Svetlana S SS Schroeter Mechthild M MM Chalovich Joseph M JM

Biophysical journal 20100901 6

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 Ca(2+)-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 ...[more]

PMID: 20858431

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Project description:BackgroundCa2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling.Methodology and principal findingsFive week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity.Conclusions/significanceGender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.

Dataset Information

Phosphorylation of caldesmon at sites between residues 627 and 642 attenuates inhibitory activity and contributes to a reduction in Ca2+-calmodulin affinity.

Publications

Phosphorylation of caldesmon at sites between residues 627 and 642 attenuates inhibitory activity and contributes to a reduction in Ca2+-calmodulin affinity.

Similar Datasets

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