Purification and preliminary characterization of an Fcε‐receptor‐activated protein‐tyrosine phosphatase from mast cells

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Abstract
Immunological stimulation of rat, mucosal type, mast cells (line RBL-2H3) by clustering the type I Fc(epsilon) receptor (Fc[epsilon]RI) causes a fast yet transient tyrosyl phosphorylation of several proteins. We report here the characterization of a protein-tyrosine phosphatase (PTP) involved in the very early steps coupling the Fc(epsilon)RI stimulus to the cell secretory response. We have observed earlier that a PTP activity present in one of the cells' solubilized particulate fractions is enhanced 2-3-fold upon cell stimulation by Fc(epsilon)RI clustering [Hampe, C. S. & Pecht, I. (1994) FEBS Lett. 346, 194-198]. This PTP (MpII PTP) was now isolated and purified to homogeneity and appears as a 45-kDa protein. Sequence comparison of two peptide stretches of MpII PTP with those of proteins present in the Swiss-Prot and EMBL data banks revealed no significant similarity to a known protein. Hence, we assume that the MpII PTP is a novel protein. The possible involvement of MpII PTP in the stimulus-secretion coupling cascade was investigated. Abrogation of the antigen-induced Fc(epsilon)RI clustering by an excess of monovalent hapten suppressed the Fc(epsilon)RI-mediated MpII PTP activity enhancement to the levels observed in resting cells. The activity enhancement required the presence of extracellular Ca2+ ions and was also induced by an artificial increase of the free intracellular concentration of these ions. It is apparently mediated by protein kinase C (PKC) since phorbol myristate induced an additive increase in the MpII PTP activity to that induced by antigen. Also, treatment with the PKC-specific inhibitor bisindolylmaleimide suppressed the antigen-induced MpII PTP activity enhancement. PKC involvement was further supported by the finding that the MpII PTP 45-kDa protein underwent seryl phosphorylation following antigen stimulation. This modification was found to be further enhanced by pretreatment with phorbol myristate prior to antigen stimulation.

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