Identification of a Novel Immunoreceptor Tyrosine-based Activation Motif-containing Molecule, STAM2, by Mass Spectrometry and Its Involvement in Growth Factor and Cytokine Receptor Signaling Pathways

Abstract

In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling molecule containing a Src homology 3 domain as well as an immunoreceptor tyrosine-based activation motif (ITAM). This molecule is 55% identical to a previously isolated molecule designated signal transducing adaptor molecule (STAM) that was identified as an interleukin (IL)-2-induced phosphoprotein and is therefore designated STAM2. Tyrosine phosphorylation of STAM2 is induced by growth factors such as epidermal growth factor and platelet-derived growth factor as well as by cytokines like IL-3. Several of the deletion mutants tested except the one containing only the amino-terminal region underwent tyrosine phosphorylation upon growth factor stimulation, implying that STAM2 is phosphorylated on several tyrosine residues. STAM2 is downstream of the Jak family of kinases since coexpression of STAM2 with Jak1 or Jak2 but not an unrelated Tec family kinase, Etk, resulted in its tyrosine phosphorylation. In contrast to epidermal growth factor receptor-induced phosphorylation, this required the ITAM domain since mutants lacking this region did not undergo tyrosine phosphorylation. Finally, overexpression of wild type STAM2 led to an increase in IL-2-mediated induction of c-Myc promoter activation indicating that it potentiates cytokine receptor signaling.A large number of cell surface receptors transmit their signals by pathways involving tyrosine phosphorylation. The receptor may themselves possess tyrosine kinase activity such as receptor protein-tyrosine kinases (e.g. epidermal growth factor receptor (EGFR) 1 ) or may not have any catalytic activity (cytokine receptors such as the erythropoietin receptor) (1-6). Signaling by cytokine receptors is mediated by cytoplasmic kinases such as the Jak or Src family of kinases (4, 7). In both of these receptors systems, a cascade of events involving tyrosine phosphorylation is initiated eventually leading to the recruitment of multiprotein complexes and activation of downstream target proteins or genes. Most of the substrates that are recruited are not specific to the receptor protein-tyrosine kinases or the cytokine receptor signaling pathways. Adapter proteins such as Shc and p85 subunit of PI 3-kinase are utilized by almost all of these signaling pathways (8, 9).Mass spectrometry-based proteomics has recently emerged as a powerful tool to analyze protein-protein interactions in general as well as in the study of receptor-mediated signaling pathways (10). We have initiated a systematic study to identify all substrates of the EGFR that are tyrosine-phosphorylated in HeLa cells. Using a combination of affinity purification using anti-phosphotyrosine antibodies and one-dimensional electrophoresis followed by mass spectrometry-based identification, we have previously reported Vav-2 as ...

Stat1-independent regulation of gene expression in response to IFN-γ

“…These two kinases are involved in many growth factor-and cytokine-dependent signaling pathways, and their essential roles have been demonstrated by gene targeting studies (47,48). Recent intensive analyses of protein expression patterns have revealed that many tyrosine-phosphorylated substrates are involved in growth factor-dependent signaling, and some of these may require Jaks for their phosphorylation (49,50). Therefore, we speculate that both Jak1 and Jak2 are involved in Stat1-independent pathways.…”

A Genetic Mosaic Analysis With a Repressible Cell Marker Screen to Identify Genes Involved in Tracheal Cell Migration During Drosophila Air Sac Morphogenesis

“…Two human Stam homologs have been described (Takeshita et al 1996;Endo et al 2000;Pandey et al 2000;Takata et al 2000). Stam proteins are constitutive binding partners of the Hepatocyte growth factor-regulated substrate (Hrs) (Asao et al 1997;Takata et al 2000).…”

Analytical strategies for phosphoproteomics

“…Single phosphoproteins can be purified from a cell lysate by immunoprecipitation using antibodies [23]. After immunoprecipitation, the sample can be separated by SDS-PAGE and the correct protein band of interest is excised and further analyzed by MS. Antibodies against phosphorylated serine, threonine, and tyrosine residues can be used for the overall enrichment of phosphoproteins from a complex sample, though, this method is limited by the specificity of the antibodies [28,29]. Tyrosine phosphorylation, in particular, is of very low abundance.…”