Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1.
The p21-activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non-muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r-MLC) in Triton-skinned guinea-pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus - MLCK, caldesmon or r-MLC - is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton-skinned carotid artery and taenia coli from guinea-pig with an active mutant of PAK1 in relaxing solution for 30-60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton-skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r-MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin-LR (10 microM), the rate of contraction and r-MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r-MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady-state force and r-MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little 32P incorporation into caldesmon. PAK1 phosphorylated isolated r-MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.
Wirth
A
A
Department of Vegetative Physiology, University of Cologne, Robert Koch-Strasse 39, D-50931 Cologne, Germany.
Schroeter
M
M
Kock-Hauser
C
C
Manser
E
E
Chalovich
J M
JM
De Lanerolle
P
P
Pfitzer
G
G
D000818:Animals; D002339:Carotid Arteries; D003106:Colon; D005260:Female; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018994:Myosin Light Chains / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000494:pharmacology*; D051381:Rats; D054462:p21-Activated Kinases
D018994:Myosin Light Chains; C515906:Pak1 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases