Localization and down-regulating role of the protein tyrosine phosphatase PTP2C in membrane ruffles of PDGF-stimulated cells.

PTP2C (also known as Syp/SH-PTP2/PTPlD) is a soluble protein tyrosine phosphatase present in most cell types. It interacts directly with activated PDGF receptor via its SH2 domains, which results in its phosphorylation on tyrosine residue(s). The phosphorylated PTP2C in turn binds to the SH2 domain of GRB2, serving as an adaptor in the transduction of mitogenic signals from the growth factor receptor to the Ras and MAP kinase signaling pathways. We investigated the interaction of PTP2C with the PDGF receptor by examining the localization of both proteins after PDGF stimulation of 293 cells which stably express the human PDGF receptor. In resting cells, transiently expressed PTP2C was distributed throughout the cytoplasm. Upon stimulation with PDGF, PTP2C was translocated from the cytoplasm to membrane ruffles. Immunofluorescence examination revealed that PTP2C colocalized with actin, the PDGF receptors, and hyper-tyrosine- phosphorylated protein(s). Neither deletion of the SH2 domains nor point mutations at either the catalytic site or the major phosphorylation site affected membrane ruffling or the localization of PTP2C to the ruffles of PDGF-stimulated cells. However, the expression of a catalytically inactive mutant PTP2C substantially prolonged ruffling activity following PDGF stimulation. These results suggest that PTP2C is involved in the down-regulation of the membrane ruffling pathway, and in contrast to its positive function in the MAP kinase pathway, the phosphatase activity negatively regulates ruffling activity.

Isolation of IFAPa-400 cDNAs: evidence for a transient cytostructural gene activity common to the precursor cells of the myogenic and the neurogenic cell lineages.

Differentiation of neural and muscle cells is characterized by a switch in the expression of the type of intermediate filament protein subunit. In these lineages, vimentin is transiently expressed in the initial stages of development and is gradually replaced by a tissue specific protein. We have identified a giant developmentally regulated antigen (IFAPa-400) which colocalizes with vimentin in the precursor cells of the neurogenic and myogenic lineages of the chick embryo [Chabot and Vincent (1990) Dev. Brain Res. 54, 195-204; Cossette and Vincent (1991) J. Cell Sci. 98, 251-260]. Based on the expression of this protein during neurogenesis and myogenesis, we hypothesize that IFAPa-400 and vimentin define a special intermediate filament network, common to the non-differentiated cells derived from the neuroectoderm and those of the myogenic tissues. We report here the isolation and sequence of partial cDNAs encoding more than 400 amino acids of the carboxy-terminus of this protein. RNA blot analysis and in situ hybridization indicate that IFAPa-400 represents a bona fide developmentally regulated gene product. These results further confirm that IFAPa-400 mRNA transcripts are limited to the early precursor cells of both neurogenic and myogenic lineages.

Expression of a developmentally regulated cross-linking intermediate filament-associated protein (IFAPa-400) during the replacement of vimentin for desmin in muscle cell differentiation.

Myogenic and neurogenic tissues of the chick embryo transiently express IFAPa-400, a high molecular weight protein that colocalizes and is copurified with intermediate filaments. Using monoclonal antibody F51H2 to identify it, we carried out immunoelectron microscopy experiments on whole-mount chick embryo cells and showed that IFAPa-400 was localized at crossing points of intermediate filaments. Also, immunoblot experiments with F51H2, anti-vimentin and anti-desmin antibodies demonstrated the complete disappearance of IFAPa-400 in those muscle cell types that change their vimentin content for desmin during embryogenesis. During in vitro myogenesis, the expression of IFAPa-400 was shown to be concurrent with the progressive replacement of vimentin by desmin in myoblasts. When long-term myotube cultures were maintained on a fibroblast-like cell layer, we observed the complete replacement of vimentin by desmin, followed by the disappearance of IFAPa-400 from the myotubes. These results suggest that IFAPa-400 might be involved in the reorganization of the intermediate filament network during muscle differentiation.