Abstract
Overexpression of a catalytically inactive mutant of tyrosine phosphatase SHP-2 in 293 cells resulted in hyperphosphorylation of a glycoprotein specifically associated with the enzyme. The protein has been purified to near homogeneity. Based on the amino acid sequences of peptides obtained from the protein, a full-length cDNA was isolated. The cDNA encodes a protein with a single transmembrane segment and a signal sequence. The extracellular portion of the protein contains a single immunoglobulin-like domain displaying 46% sequence identity to that of myelin P0, a major structural protein of peripheral myelin. The intracellular segment of the protein shows no significant sequence identity to any known protein except for two immunoreceptor tyrosine-based inhibitory motifs. We name the protein PZR for protein zero related. Transfection of the PZR cDNA in Jurkat cells gave rise to a protein of expected molecular size. Stimulation of cells with pervanadate resulted in tyrosine phosphorylation of PZR and a near-stoichiometric association of PZR with SHP-2. Northern blotting analyses revealed that PZR is widely expressed in human tissues and is particularly abundant in heart, placenta, kidney, and pancreas. As a binding protein and a putative substrate of SHP-2, PZR protein may have an important role in cell signaling.
Highlights
Protein tyrosine phosphatases (PTPs)1 represent a highly diverse family of enzymes that have a pivotal role in cell proliferation, differentiation, and transformation [1,2,3]
In human embryonic kidney 293 cells, expression of the catalytically inactive Cys-to-Ser mutant form of SHP-2 resulted in tyrosine phosphorylation of 43- and 95-kDa proteins, which were associated with SHP-2, whereas overexpression of the mutant of SHP-1 led to tyrosine phosphorylation of 95- and
Tyrosine phosphorylation of these proteins and their association with SHP-1 and/or SHP-2 were observed in cells treated with pervanadate, a potent inhibitor of PTPs
Summary
Materials—Polyclonal anti-SHP-1 and anti-SHP-2 antibodies were raised in rabbits against full-length SHP-1 and an Src homology 2 domain-truncated form of SHP-2, respectively [27, 28]. Nuclear pellets were removed by centrifugation at 800 ϫ g for 20 min, and the remaining postnuclear extract was further centrifuged at 100,000 ϫ g for 45 min to give a clear cytosolic supernatant and a pelleted membrane fraction. The eluates were loaded onto an anti-SHP-2 antibody-Sepharose column, which was equilibrated with Buffer B, washed with 0.5 M NaCl, and eluted with 2.0 M NaSCN. Throughout the purification procedure, the proteins were followed by anti-phosphotyrosine Western blot analyses.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
