Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.
Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.
Fernandez
A
A
Cell Biology Unit, Recherche Scientifique-Institut, National de la Santé et de la Recherche Médicale, Montpellier, France.
Brautigan
D L
DL
Mumby
M
M
Lamb
N J
NJ
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D008845:Microinjections; D009132:Muscles / Q000201:enzymology*; D015879:Myosin Subfragments / Q000302:isolation & purification / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000008:administration & dosage / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D015879:Myosin Subfragments; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases