Shc Proteins Are Localized on Endoplasmic Reticulum Membranes and Are Redistributed after Tyrosine Kinase Receptor Activation

Abstract

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.The Shc locus is highly conserved throughout evolution and codes for three overlapping proteins of 66, 52, and 46 kDa (p66 shc have a common carboxy-terminal SH2 domain, flanked by a glycine-and proline-rich region of approximately 145 amino acids (collagen homologous region 1 [CH1]) (38). At the N terminus, adjacent to the CH1 region, they possess a region with functional properties of a phosphotyrosine-binding domain (PTB) (4,6,24,25,47,54). The binding specificities of the Shc PTB and SH2 domains differ (3,24,44,54). The unique amino-terminal portion of p66 shc contains an additional collagen homologous region (CH2 region). The CH1 and CH2 regions are putative SH3 binding sites (34).Shc proteins are phosphorylated by all activated receptor tyrosine kinases (RTKs) tested to date: epidermal growth factor receptor (EGFR) (38, 39), platelet-derived growth factor receptor (55), hepatocyte growth factor receptor (37, 39), erbB-2 receptor (47), insulin receptor (23,26,40,41,48), fibroblast growth factor receptor (53), nerve growth factor receptor (7, 50), and sea receptor (13).

Lymphocyte Antigen Receptor Signal Integration and Regulation by the SHC Adaptor

“…This growth factor-induced association of Shc with the cytoskeleton suggests a role for Shc in membrane ruffling and actin fiber reorganization. Shc appears to be also involved in endocytosis, as it is recruited to endocytic structures such as coated pits and endosomes in response to growth factor receptor stimulation (Lotti et al, 1996). This potential role is supported by the finding of an interaction of Shc, mediated by the CH1 domain, with ␣-and ␤-adaptin components of plasma membrane adaptor proteins that are believed to be involved in receptor endocytosis (Obayashi et al, 1996).…”

Signaling via Shc family adapter proteins

“…Lotti et al (1996) also observed that Shc proteins are localized to the membranes by immuno¯uorescence. When di erent domains of Shc were transiently expressed in COS cells, the PTB domain of Shc was su cient for localization to the membrane fractions .…”

The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein

“…To address this, we investigated whether the precursor forms of RetD6 and Ret2A were able to bind the Shc adapter protein. This protein is localized to the membrane of the rough endoplasmic reticulum and has been previously demonstrated to bind Ret/ PTCs, Ret2A and Ret2B (a mutant Ret receptor carrying an M918T mutation) oncoproteins (Lotti et al, 1996). Cell lysates from RetD6 and Ret2A transfected NIH3T3 cells were immunoprecipitated with anti-Shc or anti-Ret antibodies and subsequently blotted with anti-Ptyr antibodies (Figure 3).…”