PTP1B regulates STAM phosphorylation. A, STAM2 binds ubiquitin independently of EGF-induced tyrosine phosphorylation. Lysates from myc-STAM2 (WT and 3YF)-expressing HeLa cells, untreated or stimulated with EGF for 15 min (lower panel), were subjected to in vitro PDs with GST alone or GST-tagged ubiquitin (monoubiquitin, 1×Ub, or diubiquitin, 2×Ub). Precipitated proteins were analyzed by immunoblotting (IB, upper panel). B, PTP1B knockdown augments EGF-induced tyrosine phosphorylation of GST-diubiquitin-interacting proteins. HeLa cells treated with PTP1B-targeted or scrambled siRNA were stimulated with 100 ng/ml EGF for the indicated times. Diubiquitin-interacting proteins were purified by GST PD and analyzed by immunoblotting. The positions of STAM1/2 and Hrs on the PD P-Tyr IB are indicated. TCL, total cell lysates.

Tyrosine phosphorylation of STAM2 affects its function and localization. A, tyrosine-phosphorylated STAM2 modulates EGF-induced Akt phosphorylation. HeLa cells were treated with siRNAs targeting STAM1 and STAM2 and subsequently transfected with siRNA-resistant myc-STAM2 (WT or 4YF) or mock-transfected. Control cells treated with scrambled siRNA were also mock-transfected. Cells were then stimulated with 10 ng/ml EGF for the indicated, times and total lysates were analyzed by immunoblotting. Two different exposures of the P-Akt blots are shown to avoid band saturation at early time points. B, phosphorylation-deficient STAM2 mutant displays altered localization after EGF stimulation. HeLa cells, treated with siRNAs targeting STAM1 and STAM2, were plated on coverslips and transfected with siRNA-resistant myc-STAM2 (WT or 4YF). Cells were cold-loaded with labeled EGF (100 ng/ml, red) and then transferred to 37 °C for the indicated times. Cells were then fixed, permeabilized, and stained with a myc antibody (green or white) and DAPI (blue). Results are representative of three independent experiments. Scale bar, 10 μm.

PTP1B trapping mutant binds to specific phosphotyrosine residues of STAM2. A, PTP1B D181A associates with endogenous STAM2 in intact cells. GST-PTP1B (WT or D181A) was expressed in COS-7 cells with or without c-Src. Interacting proteins were purified by GST PD and analyzed by immunoblotting (IB) along with the total cell lysates (TCL). B, STAM2/PTP1B binding site is mapped. Blots shown are as in A, except GST-PTP1B was co-expressed with myc-STAM2 (WT and YF mutants, upper panels). The results of three independent experiments were quantified (lower panel). The band density of STAM2 (myc) isolated by GST PD and in the total cell lysates were quantified by densitometry. Error bars correspond to S.E., and asterisks denote ratios significantly less than for WT STAM2 (p < 0.05, Student's t test), to which the ratios of the YF mutants were normalized. C, mutation of STAM2 Tyr²⁹¹, Tyr³⁷¹, and Tyr³⁷⁴ (3YF) blocks its interaction with PTP1B. GST-PTP1B and myc-STAM2 were expressed, and the interaction was analyzed as in B. D, PTP1B D181A and phosphorylated WT, but not 3YF, STAM2 can interact directly. Recombinant GST alone, GST-PTP1B (WT and D181A), and phosphorylated STAM2 (WT and 3YF) were purified and subjected to in vitro binding assays, and their interaction was analyzed by immunoblotting. E, STAM2 3YF displays decreased EGF-induced tyrosine phosphorylation. myc-tagged STAM2 was purified by immunoprecipitation (IP) from HeLa cells left untreated or stimulated with 100 ng/ml EGF for 15 min. F, STAM2 structure and location of PTP1B binding sites are diagrammed. STAM2 consists of N-terminal Vps27/Hrs/STAM (VHS) and ubiquitin-interacting (UI) motifs required for ubiquitin binding, a central SH3 domain, and coiled-coiled (CC), and STAM-specific (SSM) motifs implicated in Hrs binding. The positions of Tyr²⁹¹, Tyr³⁷¹, Tyr³⁷⁴, and Tyr⁴⁶¹ are indicated.