Different Molecular Mechanisms for Rho Family GTPase-dependent, Ca2+-independent Contraction of Smooth Muscle

Abstract

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca 2؉ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160 ROCK ) can induce Ca 2؉-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 ؎ 12% (n ‫؍‬ 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca 2؉ -independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.

p21-Activated Kinase 2 in Neutrophils Can Be Regulated by Phosphorylation at Multiple Sites and by a Variety of Protein Phosphatases

“…6). As reported previously, GST-Pak 3 is susceptible to degradation (33). The resulting fusion protein reacted with the pPak(AL) Ab and the pPak 1/2(Ser 144/141 ) Ab (Figs.…”

Increased Myosin Light Chain Phosphorylation Is Not Required for Growth Factor Stimulation of Collagen Matrix Contraction

“…At high concentrations of PDGF, the inhibition by PI3-kinase inhibitors appeared to be overcome, at least partially. Interestingly, smooth muscle contraction that is independent of MLC phosphorylation has been reported to involve the protein kinase PAK (33), and PAK has been implicated in cell membrane ruffling as well (34 -36). While our studies were in progress, two other laboratories also showed that blocking PI3-kinase inhibits PDGF-dependent collagen matrix contraction (37,38).…”

A GIT1/PIX/Rac/PAK Signaling Module Regulates Spine Morphogenesis and Synapse Formation through MLC

“…Because PAK directly phosphorylates MLC on serine 19 (Ramos et al, 1997;Chew et al, 1998;Van Eyk et al, 1998), we investigated the possibility that MLC acts downstream of PAK to mediate its effect on spine morphogenesis and excitatory synapse formation. We first examined the localization of activated MLC in hippocampal neurons.…”