Complex phosphatase regulation of Ca2+-activated Cl- currents in pulmonary arterial smooth muscle cells.

The present study was undertaken to determine whether the two ubiquitously expressed Ca(2+)-independent phosphatases PP1 and PP2A regulate Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) elicited by 500 nM [Ca(2+)](i) in rabbit pulmonary artery (PA) myocytes dialyzed with or without 3 mM ATP. Reverse transcription-PCR experiments revealed the expression of PP1alpha, PP1beta/delta, PP1gamma, PP2Aalpha, PP2Abeta, PP2Balpha (calcineurin (CaN) Aalpha), and PP2Bbeta (CaN Abeta) but not PP2Bgamma (CaN Agamma) in rabbit PA. Western blot and immunofluorescence experiments confirmed the presence of all three PP1 isoforms and PP2A.

Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells.

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl- currents (I(ClCa)) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, I(ClCa) was evoked immediately upon membrane rupture but then exhibited marked rundown to approximately 20% of initial values.

c-Abl is required for staurosporine-induced caspase activity.

Caspases are the intracellular molecular machinery responsible for apoptotic cell death. The regulation of these critical proteolytic enzymes is known to occur on multiple levels. While their expression as inactive precursors exhibits a primary level of control, other types of regulation such as post-translational modifications also play a role.

Oxidative stress activates both Src-kinases and their negative regulator Csk and induces phosphorylation of two targeting proteins for Csk: caveolin-1 and paxillin.

Csk negatively regulates Src family kinases (SFKs). In lymphocytes, Csk is constitutively active, and is transiently inactivated in response to extracellular stimuli, allowing activation of SFKs. In contrast, both SFKs and Csk were inactive in unstimulated mouse embryonic fibroblasts, and both were activated in response to oxidative stress.

Fyn is required for oxidative- and hyperosmotic-stress-induced tyrosine phosphorylation of caveolin-1.

Caveolin-1 is phosphorylated on Tyr(14) in response to both oxidative and hyperosmotic stress. In the present paper, we show that this phosphorylation requires activation of the Src family kinase Fyn. Stress-induced caveolin phosphorylation was abolished by three Src kinase inhibitors, SU6656, PP2 and PD180970, and was not observed in fibroblasts derived from a Src, Yes and Fyn triple-knockout mouse (SYF-/-).

c-Abl is required for oxidative stress-induced phosphorylation of caveolin-1 on tyrosine 14.

Caveolin-1 is phosphorylated at tyrosine 14 in response to cellular stress. Tyrosine 14 is a consensus Abl phosphorylation site suggesting that caveolin-1 may be an Abl substrate. We report here that expression of c-Abl is required for oxidative stress-induced caveolin-1 phosphorylation.