Regulation of EGF-induced phospholipase C-gamma1 translocation and activation by its SH2 and PH domains.
ABSTRACT Translocation of phospholipase C-gamma1 is essential for its function in response to growth factors. However, in spite of recent progress, the phospholipase C-gamma1 translocation pattern and the molecular mechanism of the translocation are far from fully understood. Contradictory results were reported as to which domain, PH or SH2, controls the epidermal growth factor-induced translocation of phospholipase C-gamma1. In this communication, we studied epidermal growth factor-induced translocation of phospholipase C-gamma1 by using comprehensive approaches including biochemistry, indirect fluorescence and live fluorescence imaging. We provided original evidence demonstrating that: (i) endogenous phospholipase C-gamma1, similar to YFP-tagged phospholipase C-gamma1, translocated to endosomes following its initial translocation from cytosol to the plasma membrane in response to epidermal growth factor; (ii) phospholipase C-gamma1 remained phosphorylated in endosomes, but phospholipase C-gamma1 activity is not required for its translocation, which suggests a signaling role for phospholipase C-gamma1 in endosomes; (iii) the PH domain was not required for the initial translocation of phospholipase C-gamma1 from cytosol to the plasma membrane, but it stabilizes phospholipase C-gamma1 in the membrane at a later time; (iv) the function of the phospholipase C-gamma1 PH domain in stabilizing phospholipase C-gamma1 membrane association is very important in maintaining the activity of phospholipase C-gamma1; and (v) the role of the PH domain in phospholipase C-gamma1 membrane association and activation is dependent on PI3K activity. We conclude that the phospholipase C-gamma1 SH2 and PH domains coordinate to determine epidermal growth factor-induced translocation and activation of phospholipase C-gamma1.
Article: Akt binds to and phosphorylates phospholipase C-gamma1 in response to epidermal growth factor.[show abstract] [hide abstract]
ABSTRACT: Both phospholipase (PL) C-gamma1 and Akt (protein kinase B; PKB) are signaling proteins that play significant roles in the intracellular signaling mechanism used by receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR). EGFR activates PLC-gamma1 directly and activates Akt indirectly through phosphatidylinositol 3-kinase (PI3K). Many studies have shown that the PLC-gamma1 pathway and PI3K-Akt pathway interact with each other. However, it is not known whether PLC-gamma1 binds to Akt directly. In this communication, we identified a novel interaction between PLC-gamma1 and Akt. We demonstrated that the interaction is mediated by the binding of PLC-gamma1 Src homology (SH) 3 domain to Akt proline-rich motifs. We also provide a novel model to depict how the interaction between PLC-gamma1 SH3 domain and Akt proline-rich motifs is dependent on EGF stimulation. In this model, phosphorylation of PLC-gamma1 Y783 by EGF causes the conformational change of PLC-gamma1 to allow the interaction of its SH3 domain with Akt proline-rich motifs. Furthermore, we showed that the interaction between PLC-gamma1 and Akt resulted in the phosphorylation of PLC-gamma1 S1248 by Akt. Finally, we showed that the interaction between PLC-gamma1 and Akt enhanced EGF-stimulated cell motility.Molecular Biology of the Cell 06/2006; 17(5):2267-77. · 4.94 Impact Factor
Article: An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family.[show abstract] [hide abstract]
ABSTRACT: We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca(2+)] that can activate calmodulin. We postulate the Ca(2+)/calmodulin complex binds rapidly to residues 645--660 of the juxtamembrane domain, reversing its net charge from +8 to -8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP(2)), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca(2+)/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP(2); and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.The Journal of General Physiology 08/2005; 126(1):41-53. · 3.84 Impact Factor
Article: Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis.[show abstract] [hide abstract]
ABSTRACT: Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, we have generated cell lines in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl did not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis was rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The "knockdown and rescue" analysis revealed that the expression of Cbl-Grb2/SH2 fusions containing RING finger domain of Cbl restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cbl, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cbl carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. We concluded that Grb2-mediated recruitment of the functional RING domain of Cbl to the EGFR is essential and sufficient to support receptor endocytosis.Molecular Biology of the Cell 04/2005; 16(3):1268-81. · 4.94 Impact Factor