Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase.
Microinjection of the catalytic subunit of cAMP-dependent protein kinase (A-kinase) into living fibroblasts or the treatment of these cells with agents that elevate the intracellular cAMP level caused marked alterations in cell morphology including a rounded phenotype and a complete loss of actin microfilament bundles. These effects were transient and fully reversible. Two-dimensional gel electrophoresis was used to analyze the changes in phosphoproteins from cells injected with A-kinase. These experiments showed that accompanying the disassembly of actin microfilaments, phosphorylation of myosin light chain kinase (MLCK) increased and concomitantly, the phosphorylation of myosin P-light chain decreased. Moreover, inhibiting MLCK activity via microinjection of affinity-purified antibodies specific to native MLCK caused a complete loss of microfilament bundle integrity and a decrease in myosin P-light chain phosphorylation, similar to that seen after injection of A-kinase. These data support the idea that A-kinase may regulate microfilament integrity through the phosphorylation and inhibition of MLCK activity in nonmuscle cells.
Lamb
N J
NJ
Cold Spring Harbor Laboratory, New York 11724.
Fernandez
A
A
Conti
M A
MA
Adelstein
R
R
Glass
D B
DB
Welch
W J
WJ
Feramisco
J R
JR
D008841:Actin Cytoskeleton / Q000502:physiology*; D000199:Actins / Q000502:physiology*; D000818:Animals; D003599:Cytoskeleton / Q000502:physiology*; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008845:Microinjections; D008854:Microscopy, Electron; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D009218:Myosins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D051381:Rats; D013997:Time Factors
D000199:Actins; D010750:Phosphoproteins; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D009218:Myosins