Abstract
Four different forms of a non-receptor type protein-tyrosine phosphatase are generated by alternative splicing; two of these forms (PTP-S2 and PTP-S4) are major forms, which are expressed in rat as well as human cells. Here we report that PTP-S2 binds to nonspecific DNA in vitro and localizes in the nucleus upon transfection in HeLa cells. PTP-S4 does not bind to nonspecific DNA and shows perinuclear and cytoplasmic localization. Removal of the C-terminal 34 amino acids of PTP-S4 gives rise to a truncated protein, which binds to nonspecific DNA and localizes to the nucleus. PTP-S4, but not PTP-S2, interacts strongly with the isolated nuclear matrix. The two forms of this tyrosine phosphatase show different substrate specificity in vitro, a feature novel to splice variants of tyrosine phosphatases. Mitogenic stimulation induces mRNAs for PTP-S2 as well as for PTP-S4 in the G1 phase during liver regeneration. These results suggest that alternative splicing gives rise to two protein-tyrosine phosphatases with distinct substrate specificities and subcellular locations. The 34 amino acids at the C terminus of PTP-S4 play a critical role in determining substrate specificity, subcellular location, and interaction with nuclear matrix and DNA.
Highlights
The cloning and characterization of several genes encoding protein-tyrosine phosphatases (PTPs)1 has drawn attention to their significance in regulating various cellular processes by modulating the level of phosphotyrosine in the cell
We have recently shown that four different forms of PTP-S, which arise due to alternative splicing [19], are expressed in rat cells
The C-terminal 6 amino acids of PTP-S2 do not appear to be critical for interaction with DNA, they contribute to some extent to this interaction (Fig. 1, B and C, lanes 2 and 4)
Summary
Construction of Expression Vectors—PTP-S2 and PTP-S4 cDNAs, described previously, were cloned by amplifying mRNA using reverse transcription and PCR [19] These were cloned in the BamHI site of. The resulting cDNA was digested with BamHI, cloned in pUC18, sequenced, and cloned in pET-3a This construct named ⌬PTP-S4 gives rise to a protein lacking the C-terminal 34 amino acids of PTP-S4 (Fig. 1D). These cDNAs were cloned in pCB6 vector in the BglII site for expressing in eukaryotic cells. In each reaction during amplification, 2 Ci of [␣-32P]dATP was included
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