Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

Abstract

Abstract. 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 rain) 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 afffinity-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. EVERSIBLE protein phosphorylation is well known to play an implicit role in the mechanisms that regulate actomyosin contractility (1,(18)(19)(20)27; reviewed in references 32, 43). There has been a consensus that the increased phosphorylation of the 20-22-kD myosin light chain component of the myosin complex coincides with the increased contractility of smooth and skeletal muscle tissue (2,5,19,20,41; reviewed in references 2, 27, 32, 43), and is related to changes in actin organization and cell shape in nonmuscle cells (6,33). A calcium/calmodulin-dependent enzyme, myosin light chain kinase (MLCK), 1 catalyzes this reaction (3, 17; reviewed in references 32, 43). More controversial has been understanding the mechanisms which bring about the regulation of this enzyme in living muscle cells, since in addition to its activation by calmodulin (17; reviewed in reference 32), MLCK activity can be modulated through direct phosphorylation by the cAMP-dependent pro- tein kinase (A-kinase) (3, 17, 28; reviewed in references 32, 43). We have recently shown that inhibition of MLCK by microinjection of anti-MLCK antibodies into the mammalian cell line REF-52 led to the dephosphorylation of myosin light chain, and the disassembly of the actin microfilam...

Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55

“…In contrast, TAU treatment led to increased staining for phalloidin, indicative of enhanced polymerization of G-to F-actin. Again this observation is in agreement with the reported role of PP1 in dephosphorylating myosin light chain (Fernandez et al, 1990). Vimentin from TAUtreated cells underwent limited reorganization, but in contrast to OA, no thick bundles were observed.…”

“…substrate for PP1 in fibroblasts (Fernandez et al, 1990). OA treatment rather induced a diminution of myosin light chain phosphorylation, which may be attributable to a role of PP2A in regulating myosin light chain kinase.…”

“…The remaining material was found colocalized with vimentin IF. Using a similar pre-extraction technique, our laboratory has previously visualized the association of PP1 with actin microfilaments (Fernandez et al, 1990). Immunochemical analysis of purified vimentin, isolated under highly stringent conditions, further corroborated a physical association of PP2A 1 with vimentin.…”

“…Vimentin was heavily hyperphosphorylated in cells treated with OA when compared with that of control or TAUtreated cells ( Figure 1A, upper panel). In contrast, TAU treatment led to hyperphosphorylation of myosin light chain ( Figure 1A, lower panel), a bona fide substrate for PP1 in fibroblasts (Fernandez et al, 1990). OA treatment rather induced a diminution of myosin light chain phosphorylation, which may be attributable to a role of PP2A in regulating myosin light chain kinase.…”

Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines

“…Spine density and morphology have been shown to be dynamic during development and in the adult brain (1) and the requirement for a dynamic regulation of spine anatomy may explain the presence of the dense actin microfilament network within the cytoskeleton of spine heads. Previous work in fibroblasts has shown that actin microfilament assemblies are regulated by the PP1-mediated dephosphorylation of myosin light chain (19). Thus, an important function of spinophilin, in addition to regulating the state of phosphorylation and activity of ion channels and neurotransmitter receptors, might be to target PP1 to the spine microfilament network to regulate spine number and͞or morphology.…”