Endocrinology, Vol 133, 2838-2846, Copyright © 1993 by Endocrine Society

ARTICLES

Evidence for the potentiation of epidermal growth factor receptor tyrosine kinase activity by association with the detergent-insoluble cellular cytoskeleton: analysis of intact and carboxy-terminally truncated receptors

AM Gronowski and PJ Bertics
Endocrinology-Reproductive Physiology Program, University of Wisconsin- Madison 53706.

The ligand-stimulated tyrosine kinase activity of the normal human epidermal growth factor (EGF) receptor and a truncated EGF receptor lacking 164 carboxy-terminal (C-terminal) amino acids was examined in intact cells and after Triton X-100 extraction into Triton-soluble and - insoluble (cytoskeletal) preparations. Detergent extraction of the intact and truncated receptors appeared complete using 0.3% Triton as demonstrated by anti-EGF receptor immunoblots, tyrosine kinase assays, and marker enzyme (alkaline phosphatase) solubilization. Higher Triton concentrations yielded no additional EGF receptor extraction and began to inhibit EGF-stimulated kinase activity toward angiotensin II (AII). Furthermore, the tyrosine kinase activity of the truncated EGF receptor exhibited increased sensitivity to Triton extraction, suggesting a lower affinity or a more labile association of this receptor with the cytoskeleton. However, both EGF receptor forms had altered catalytic activity when associated with the cytoskeletal fraction, as evidenced by the increased phosphorylation of the exogenous substrates: AII, src- peptide, and [Val5]AII. Kinetic analyses of both receptor types revealed that the cytoskeletal fractions obtained using 0.3% Triton contain EGF receptor activity that exhibits a Michaelis-Menten constant (Km) for AII that is 2- to 3-fold more favorable than that calculated for the soluble receptor forms. EGF treatment of intact cells containing either the intact or truncated receptor revealed similar phosphorylated proteins in the soluble fraction of both cell types, although there was evidence for the enhanced phosphorylation of certain proteins (e.g. 115 and 50 kilodalton proteins) in cells containing the truncated receptor. There was also a greater number of tyrosine- phosphorylated proteins in the Triton-insoluble fraction of cells containing the truncated receptor, suggesting an altered specificity of this receptor toward selected cytoskeletal proteins. This work indicates that EGF receptor-cytoskeletal interaction may be an important consideration in the control of receptor-kinase activity and has examined the detergent sensitivity of this association. These studies also suggest that the C-terminal domain of the EGF receptor may affect cytoskeletal interaction in addition to influencing the receptor's catalytic capacity.

This article has been cited by other articles:

				G. Orr, D. Hu, S. Ozcelik, L. K. Opresko, H. S. Wiley, and S. D. Colson Cholesterol Dictates the Freedom of EGF Receptors and HER2 in the Plane of the Membrane Biophys. J., August 1, 2005; 89(2): 1362 - 1373. [Abstract] [Full Text] [PDF]

				R. Brock and T. M. Jovin Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation J. Cell Sci., January 7, 2001; 114(13): 2437 - 2447. [Abstract] [Full Text] [PDF]

				Y.-N. Kim, G. J. Wiepz, A. G. Guadarrama, and P. J. Bertics Epidermal Growth Factor-stimulated Tyrosine Phosphorylation of Caveolin-1. ENHANCED CAVEOLIN-1 TYROSINE PHOSPHORYLATION FOLLOWING ABERRANT EPIDERMAL GROWTH FACTOR RECEPTOR STATUS J. Biol. Chem., March 10, 2000; 275(11): 7481 - 7491. [Abstract] [Full Text] [PDF]

				J. Li, Y.-N. Kim, and P. J. Bertics Platelet-derived Growth Factor-stimulated Migration of Murine Fibroblasts Is Associated with Epidermal Growth Factor Receptor Expression and Tyrosine Phosphorylation J. Biol. Chem., January 28, 2000; 275(4): 2951 - 2958. [Abstract] [Full Text] [PDF]

				J. Li, M.-L. Lin, G. J. Wiepz, A. G. Guadarrama, and P. J. Bertics Integrin-mediated Migration of Murine B82L Fibroblasts Is Dependent on the Expression of an Intact Epidermal Growth Factor Receptor J. Biol. Chem., April 16, 1999; 274(16): 11209 - 11219. [Abstract] [Full Text] [PDF]

				P Nagy, A Jenei, A. Kirsch, J Szollosi, S Damjanovich, and T. Jovin Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy J. Cell Sci., January 6, 1999; 112(11): 1733 - 1741. [Abstract] [PDF]

				M. Silvy, P.-M. Martin, N. Chajry, and Y. Berthois Differential Dose-Dependent Effects of Epidermal Growth Factor on Gene Expression in A431 Cells: Evidence for a Signal Transduction Pathway That Can Bypass Raf-1 Activation Endocrinology, May 1, 1998; 139(5): 2382 - 2391. [Abstract] [Full Text] [PDF]

				M. E. Bates, W. W. Busse, and P. J. Bertics Interleukin 5 Signals through Shc and Grb2 in Human Eosinophils Am. J. Respir. Cell Mol. Biol., January 1, 1998; 18(1): 75 - 83. [Abstract] [Full Text]

				L. J. McCawley, P. O'Brien, and L. G. Hudson Overexpression of the Epidermal Growth Factor Receptor Contributes to Enhanced Ligand-Mediated Motility in Keratinocyte Cell Lines Endocrinology, January 1, 1997; 138(1): 121 - 127. [Abstract] [Full Text] [PDF]

				S. Caplan and M. Baniyash Normal T Cells Express Two T Cell Antigen Receptor Populations, One of Which Is Linked to the Cytoskeleton via zeta Chain and Displays a Unique Activation-dependent Phosphorylation Pattern J. Biol. Chem., August 23, 1996; 271(34): 20705 - 20712. [Abstract] [Full Text] [PDF]

				M Diakonova, B Payrastre, A. van Velzen, W. Hage, P. van Bergen en Henegouwen, J Boonstra, F. Cremers, and B. Humbel Epidermal growth factor induces rapid and transient association of phospholipase C-gamma 1 with EGF-receptor and filamentous actin at membrane ruffles of A431 cells J. Cell Sci., January 6, 1995; 108(6): 2499 - 2509. [Abstract] [PDF]