Abstract
src homology region 2 and 3 (SH2 and SH3) domains are conserved noncatalytic regions originally described in cytoplasmic tyrosine kinases and subsequently identified in phospholipase C gamma 1 (PLC gamma 1), GTPase-activating protein of ras, and other signaling proteins. Although numerous studies indicate that SH2 domains promote protein-protein interactions by specific binding to tyrosine phosphorylated proteins, the function of SH3 domains is not known. The SH2 domain of PLC gamma 1 binds to certain tyrosine-phosphorylated growth factor receptors, and following phosphorylation on Tyr783 the enzymatic activity of PLC gamma 1 is enhanced, leading to phosphatidylinositol hydrolysis. To determine the functional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in Escherichia coli PLC gamma 1 constructs encoding the region containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjected to quantitative phosphorylation by the epidermal growth factor receptor (EGFR). Analysis of the kinetics of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanced for substrates containing SH2 domains: from S0.5 (average apparent Km) of 110 microM to S0.5 of 20 microM with the addition of a single SH2 domain and S0.5 of 3-4 microM for mutants containing two SH2 domains. The presence of the SH3 domain did not influence the apparent Km of substrate phosphorylation. These results demonstrate that the presence of the SH2 domain in PLC gamma 1 lowers the apparent Km (increases the affinity) of substrate phosphorylation by the EGFR, thereby facilitating PLC gamma 1 phosphorylation and activation.
Highlights
From the $Department of Pharmacology, New York University Medical Center, New York, New York10016 and the
The presenceof the SH3 domain did not influence the apparentK,of substrate phosphorylation. These results demonstrate that the presence of the src homology region2 (SH2) domain inPLCr,lowerstheapparent K, substrate phosphorylation by phosphorylated growth factor receptors
In this report we show that the presence of the SH2 domains of PLCyl limits the kinetics of substrate phosphorylationby the EGFR
Summary
From the $Department of Pharmacology, New York University Medical Center, New York, New York10016 and the. 81%homology region 2 and 3 (SH2 and SH3) domains Binding of epidermal growth factor (EGF)' to its receptor a re conservednoncatalyticregionsoriginally de- (EFGR) leads to receptor dimerization, activation of the scribedin cytoplasmic tyrosinekinasesand subse- tyrosine kinase function, and subsequent intermolecular auquentlyidentified in phospholipase Cr(lPLCrl), tophosphorylation on several tyrosine residues located at the GTPase-activating proteinof r w , and other signaling carboxyl-terminal tail of the receptor H2 and SH3 domains are conserved domain, and five different deletion mutants of this noncatalytic regions originally described in cytoplasmic tyregion. These six proteinswere purified andsubjected rosine kinases such as pp60""'" (Sadowski et al, 1986) and toquantitative phosphorylationby theepidermal subsequently found in other proteins, including PLCyl (Suh growth factor receptor(EGFR). The affinity was enhanced for subsctornat-es 3-kinase-associated p85 (Escobedo et al, 1991; Otsu et al, taining SH2 domains: from So.s (average apparentK,,,) 1991;Skolnik et al, 1991).The SH2 domains of these proteins of 110 PM to So. of 20 M M with the additionof a single were shown to be responsible for their binding to tyrosine-
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