Cloning and characterization of two protein tyrosine phosphatases (PTPases) from embryonic and adult murine sources.

Title: Cloning and characterization of two protein tyrosine phosphatases (PTPases) from embryonic and adult murine sources.
Authors: Marshall, Trudy D.
Date: 1994
Abstract: A PCR strategy was used to amplify novel sequences residing between two degenerate protein tyrosine phosphatase (PTPase) -specific primers. Partial Class I and II clones were isolated from a library constructed with this P19 stem cell derived-cDNA. The predicted amino acid sequence of these PCR fragments revealed sufficient identity with the PTPase consensus to indicate that a region from two individual PTPases had been cloned. Northern analysis of the Class II or PTP2 PTPase revealed a 5 Kb mRNA in all mouse tissues and cell lines examined, while the mouse CAP-1 (Class I) gene was found to be expressed as 5 and 7 Kb mRNA transcripts in a variety of mouse tissues. CAP-1 PTPase cDNAs have been cloned from messenger RNA derived from mouse kidney and embryonal carcinoma cells. The composite cDNA contains an open reading frame which encodes 942 amino acids. The N-terminal portion of the predicted protein (M$\sb{\rm r}$ = 107,000) shares 30-37% identity with homologous regions in the cytoskeletal associated proteins, band 4.1, ezrin, moesin, radixin, and talin. This PTPase has been termed CAP-1 for cytoskeletal associated protein related PTPase. The C-terminal phosphatase domain is 33-41% identical to catalytic domains of known PTPases. The bacterially expressed GST/CAP-1 fusion protein preferentially removes phosphate moieties from tyrosine residues. CAP-1 exhibits a specific pattern of transcription in several tissues, that is, in leg muscle as well as heart and brain. In adult leg muscle, CAP-1 is expressed as two distinct transcripts which are approximately 500 bp shorter than that found in other murine tissues. The corresponding skeletal muscle cDNAs were cloned to elucidate the mechanism by which the different mRNAs arise. The composite leg muscle CAP-1 (LMCAP-1) cDNA, with leg muscle-specific sequence at the 5$\sp\prime$ end, predicts a truncated LMCAP-1 protein which lacks part of the protein 4.1 homology region. This CAP-1myc fusion was transiently expressed in COS-1 cells to elucidate the localization and possible function of CAP-1. A CAP-1myc specific protein close to the predicted molecular weight (110-120 kDa) was immunoprecipitated from these metabolically labeled cells. Concurrently, indirect immunofluorescence with an $\alpha$myc antibody revealed a punctate, non-nuclear pattern of exogenous CAP-1myc expression in transfected COS-1 cells. Morphological changes such as the "giant cells" observed in several CAP-1myc/pMT21- and CAP-1myc/pECE-generated NIH 3T3 clones, appear unique to CAP-1, suggesting that this phenomenon is a result of the dephosphorylation of a cytoskeleton/membrane protein found normally associated with this enzyme.
CollectionThèses, 1910 - 2010 // Theses, 1910 - 2010
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