Cloning, bacterial expression, purification and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase
Abstract
Protein tyrosine phosphorylation has been determined over the past decade to be critical in the regulation of cell growth and differentiation, causing unrestrained tissue expansion and cancer when uncontrolled. Enzymes that cause the phosphorylation of tyrosine were the first of this regulatory shuttle to be defined structurally, through the examination of oncogenes. The character of the dephosphorylating enzymes, protein tyrosine phosphatases (PTPases), has only recently been determined. To further understand the mechanism by which these PTPases function, a receptor-like PTPase from rat brain, rLAR, was cloned and its cytoplasmic phosphatase domain was expressed to high levels in bacteria. Purification of this enzyme by three chromatographic steps to a single band on SDS-PAGE allowed its properties to be examined free of interfering contaminants. rLAR specifically dephosphorylated phosphotyrosine containing substrates and had behaviors similar to PTPases isolated from natural sources. Site-directed mutagenesis of four highly conserved cysteine residues in the two duplicated domains of the protein showed that greater than 99% of the enzymatic activity, on the substrates tested, resides in the first domain. The unique behavior of iodoacetate versus iodoacetamide in inhibiting phosphatase activity is consistent with a positively charged substrate binding pocket. Formation of a phosphocysteine enzyme intermediate dependant on the presence of the catalytically essential cysteine 1522 suggests that the enzyme uses a two step mechanism of catalysis, with the second step being rate limiting. Other kinetic data support this hypothesis. Phosphorylation of pure rLAR in vitro to high stoichiometries by protein kinase C and p43$\sp{\rm v-abl}$ on serine/threonine and tyrosine respectively, suggests a potential regulatory mechanism for this receptor-like PTPase.
Degree
Ph.D.
Advisors
Dixon, Purdue University.
Subject Area
Molecular biology
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