PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeleton-associated proteins. Here, we report the purification and biochemical characterization of this enzyme from baculovirus-infected insect cells. The purified protein exhibited an apparent M(r) of 120,000 on SDS gels. The native enzyme dephosphorylated both myelin basic protein (MBP) and reduced, carboxamidomethylated, and maleylated lysozyme (RCML) but was over 5-fold more active on MBP. The Km values for the two substrates were similar (1.45 microM for MBP and 1.6 microM for RCML). Phosphorylation of PTPH1 by protein kinase C in vitro resulted in a decrease in Km but had no effect on Vmax. Removal of the NH2-terminal band 4.1 homology domain of PTPH1 by limited trypsin cleavage stimulated dephosphorylation of RCML but inhibited its activity toward MBP. The dephosphorylation of RCML by full-length PTPH1 was enhanced up to 6-fold by unphosphorylated MBP and increasing ionic strength up to 0.2 M NaCl, whereas trypsinized preparations of PTPH1 containing the isolated catalytic domain were unaffected. These results suggest that in addition to a potential role in controlling subcellular localization, the NH2-terminal band 4.1 homology domain of PTPH1 may exert a direct effect on catalytic function.

Transient waves of Ca2+ release cross-fertilizing deuterostome eggs from the point of sperm entry to its antipode and provide much of the activating stimulus for the egg. Based on several indirect lines of experimental evidence, it was proposed that protostome eggs are activated by a prolonged uptake of Ca2+ from the medium due to sperm-induced membrane depolarization and that this uptake then starts an activation wave similar to those in deuterostomes, except that it moves inward from the whole surface rather than through the egg from pole to pole. To test these hypotheses, we microinjected oocytes of the polychaete annelid, Chaetopterus pergamentaceus, with semisynthetic recombinant aequorins and measured light emission in response to both fertilization and artificial activation by excess K+. Both fertilization and K(+)-activation induced multiple, brief Ca2+ transients in the eggs. The first transient did not propagate, but it was followed by a series of globally propagated Ca2+ waves interspersed with additional nonpropagated pulses. The waves traversed the egg at about 30 micrometer/sec. Sequential propagated waves and nonpropagated pulses generally originated at different regions of the egg surface, except the last few, which originated in the same "pacemaker" region. These new data are consistent with the hypothesis that the activation of protostome eggs is initiated by Ca2+ waves. However, the fact that these waves propagated from pole to pole like those in deuterostome eggs refutes the notion that Ca2+ waves in activating protostome eggs move inward from the whole surface.

In frog oocytes, activation of mitogen-activated protein kinase (MAPK, ERK) leads to activation of cdc2 and germinal vesicle breakdown (GVBD). By contrast, in starfish, MAPK is activated after GVBD. Here we have examined the relative involvements of MAPK and cdc2 in GVBD of Chaetopterus oocytes. MAPK was rapidly tyrosine-phosphorylated and activated (within 1-2 min) in response to exposure of the oocytes either to natural seawater (the normal trigger of GVBD in this organism) or to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which can also elicit GVBD. This response preceded the tyrosine dephosphorylation and activation of cdc2 by several minutes. MAPK phosphorylation and activation were transient, lasting only until GVBD occurred and the spindle migrated to the cortex. The enzyme was not phosphorylated again as a result of egg activation. These results are consistent with the hypothesis that the activation of MAPK has a role in GVBD. However, PD 98059, a potent and selective inhibitor of MEK, the protein kinase that phosphorylates and activates MAPK, blocked the phosphorylation of MAPK but did not block GVBD, the dephosphorylation and activation of cdc2, or spindle formation and migration. Oocytes that underwent GVBD in PD 98059 could be fertilized and cleaved normally. Ionophore A23187, although it caused germinal vesicles to disappear and caused transient phosphorylation of MAPK, did not cause dephosphorylation of cdc2, and therefore this disappearance is artifactual. These results suggest that MAPK activation is neither obligatory nor sufficient for either GVBD or meiotic metaphase arrest in Chaetopterus and that activation of MAPK and cdc2 occur on independent, parallel pathways.