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A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma
M C Maa1, T H Leu, B J Trandel
1Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Abstract:
p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.
Insights
A protein known as p62 associates with both GTPase-activating protein (GAP) and phospholipase C gamma (PLC gamma). This study shows p62 directly binds PLC gamma, suggesting a link between signaling pathways.
Area of Science:
- Cellular signaling
- Protein-protein interactions
- Biochemistry
Background:
- p62 protein was initially identified in association with GTPase-activating protein (GAP) of p21ras.
- Tyrosine kinase signaling pathways are crucial in cell growth and transformation.
Purpose of the Study:
- To investigate the relationship between p62, GAP, and phospholipase C gamma (PLC gamma).
- To determine if p62 directly interacts with PLC gamma and to elucidate the binding mechanism.
Main Methods:
- Immunoprecipitation assays to detect protein-protein interactions.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and comparison.
- Phosphotryptic peptide mapping to assess protein similarity.
- In vitro binding assays using glutathione S-transferase (GST) fusion proteins.
Main Results:
- A 62-kDa protein, highly related to GAP-associated p62, was found to associate with PLC gamma in response to epidermal growth factor (EGF) or v-src transformation.
- p62 and PLC gamma association was direct, not mediated by GAP or the EGF receptor.
- p62 binding to PLC gamma required both N- and C-terminal Src homology 2 (SH2) domains of PLC gamma, unlike GAP which uses its N-terminal SH2 domain.
Conclusions:
- A protein identical or highly related to GAP-associated p62 directly binds to PLC gamma.
- This interaction suggests a mechanism for cross-talk between PLC gamma-mediated and GAP-mediated signaling pathways.