It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.
Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubuleassociated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration.Mitogen-activated protein kinase (MAPK), also known as the extracellular signal-regulated kinase (ERK), is involved in the transmission of signals between plasma membrane receptors and the nucleus (1, 2). MAPK has been shown to phosphorylate and regulate cytoskeletal components such as the microtubule-associated proteins in vitro, and it was originally named microtubule-associated protein-2 kinase after its substrate, MAP2 (3). Nonetheless, numerous immunocytochemical analyses have failed to show an association of MAPK with microtubules in proliferating cells (4-9). Thus, despite the often misunderstood nature of the original name, it is generally considered that MAPK is not actually associated with the microtubules in systems where mitogenesis occurs. However, MAPK was shown to associate with microtubules in certain rat brain dendrites in situ (10) and to copolymerize with bovine brain microtubules in vitro (11). MAPK was also shown to associate with the microtubule-organizing centers, but not spindle structures, in mouse oocytes (12). These findings raise the possibility that MAPK physically interacts with and regulates microtubule dynamics under certain unique circumstances such as meiosis and dendritic remodeling in the brain.In proliferating cells, significant evidence suggests that MAPK plays a role in cytoskeletal regulation. In addition to MAPs found only in the brain, such as MAP2 and tau, MAPK also phosphorylates cytoskeletal components present in cycling cells such as MAP4 and caldesmon (13,14). MAPs, which bind to and stabilize microtubules, are phosphorylated in response to cell stimulation by a variety of mitogens. The resulting phosphorylation inhibits their capacity to stabilize the microtubules (15). MAPK, which is activated by these mitogens, has been shown to be causative in MAP inhibition in vitro (13,16). MAPK activation is triggered not only by a large number of mitogens but also upon integrin-extracellular matrix association (17, 18), a first step toward cell spreading. Although these findings collectively imply a possible role for MAPK in the regulation of the cytoskeleton, the abovedescribed immunofluorescence evidence sugge...
PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.
Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in signal transduction. The regulatory role of protein-tyrosine phosphatases (PTPs) in this process was explored by studying the effects of a powerful PTP inhibitor, pervanadate, on the activation of the mitogen-activated protein (MAP) kinase cascade. Treatment of HeLa cells with pervanadate resulted in a marked inhibition of PTP activity, accompanied by a drastic increase in tyrosine phosphorylation of cellular proteins. The increased tyrosine phosphorylation coincided with the activation of the MAP kinase cascade as indicated by enzymatic activity assays of MEK (MAP kinase/ERK-kinase) and MAP kinase and gel mobility shift analyses of Raf-1 and MAP kinase. The activation was sustained but reversible. Upon removal of pervanadate, both tyrosine phosphorylation and MAP kinase activation declined to basal levels. Therefore, inhibition of PTP activity is sufficient per se to initiate a complete MAP kinase activation program.
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