Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction.
We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.
Foster
D B
DB
Department of Biochemistry, Queen's University, Kingston Ontario, Canada K7L 3N6.
Shen
L H
LH
Kelly
J
J
Thibault
P
P
Van Eyk
J E
JE
Mak
A S
AS
D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000037:antagonists & inhibitors; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002645:Chickens / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005895:Gizzard, Avian / Q000201:enzymology / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013997:Time Factors; D014335:Tropomyosin / Q000378:metabolism; D054462:p21-Activated Kinases
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D014335:Tropomyosin; D000205:Actomyosin; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D000251:Adenosine Triphosphatases; D002118:Calcium