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

doi:10.1074/jbc.273.36.23433

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

From the Departments of ^‡Biochemistry and ^§Physiology, Queen’s University, Kingston, Ontario K7L 3N6, Canada and the ^‖New York College of Osteopathic Medicine of the New York Institute of Technology, Old Westbury, New York 11542

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 Ca²⁺ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160^ROCK) can induce Ca²⁺-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 Ca²⁺-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.

Footnotes

↵* This work was funded by the Ontario Heart and Stroke Foundation and the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ A Canadian Heart and Stroke Scholar. To whom correspondence should be addressed. Tel.: 613-545-6535; Fax: 613-545-6880; E-mail:JVE1{at}post.queensu.ca.
↵2 R. Cerione, personal communication.
Abbreviations:

MLCK

myosin light chain kinase

MRLC

20-kDa myosin regulatory light chain

PAK

p21-activated protein kinase

MLCP

myosin light chain phosphatase

ROK

Rho-associated kinase

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

MES

4-morpholineethanesulfonic acid

GTPγS

guanosine 5′-O-(thiotriphosphate).
- Received May 26, 1998.
- Revision received July 6, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.