Two Splice Variants of a Tyrosine Phosphatase Differ in Substrate Specificity, DNA Binding, and Subcellular Location

Abstract

Four different forms of a non-receptor type proteintyrosine 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 G 1 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.

Physical and Functional Interaction between Hck Tyrosine Kinase and Guanine Nucleotide Exchange Factor C3G Results in Apoptosis, Which Is Independent of C3G Catalytic Domain

“…After this the blot was washed in TBSTG for 15 min (3 washes each for 5 min). The blot was then processed with primary antibody against SH3 domain of Hck followed by secondary antibody as described for Western blotting (26,31). Detection of primary antibodies was either by ECL or by color development using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate for alkaline phosphatase-conjugated secondary antibodies.…”

“…THP-1 human myelomonocytic cell line was grown in RPMI with 10% heatinactivated fetal calf serum. Transient transfections were done using Qiagen column-purified plasmids and LipofectAMINE reagent (Invitrogen) according to the manufacturer's protocol and as described previously (31). For immunofluorescence staining, cells grown on coverslips were transfected with the required plasmids, fixed after 30 -42 h, and stained with the required antibodies as described earlier (31).…”

“…Transient transfections were done using Qiagen column-purified plasmids and LipofectAMINE reagent (Invitrogen) according to the manufacturer's protocol and as described previously (31). For immunofluorescence staining, cells grown on coverslips were transfected with the required plasmids, fixed after 30 -42 h, and stained with the required antibodies as described earlier (31). Dual labeling for Hck and C3G was performed by incubating the cells serially with C3G antibody, anti-rabbit Cy3, mouse monoclonal anti-Hck, and anti-mouse fluorescein isothiocyanate.…”

Caspase-1 activator Ipaf is a p53-inducible gene involved in apoptosis

“…Cells grown on coverslips were transfected with the required plasmids and fixed after 30 h (or later as indicated) and stained with required antibodies as described earlier (Kamatkar et al, 1996). p53 antibody was from Roche Molecular Biochemicals.…”

“…The primers used for amplifying Ipaf were 5 0 -CTC TCA TGG TGG AAG CCA GTCC-3 0 and 5 0 -GAC AGA GAC TTG ACT ATG TAA TCC-3 0 . Primers for caspase-1 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) have been described previously (Kamatkar et al, 1996;Gupta et al, 2001). Appropriate gene-specific primers were used for amplification of PUMA, Apaf-1, p53 and p21.…”