Abstract
Phospholipase Cepsilon (PLCepsilon) is activated by various growth factors or G-protein-coupled receptor ligands via different activation mechanisms. The Ras association (RA) domain of PLCepsilon is known to be important for its ability to bind with Ras-family GTPase upon growth factor stimulation. In the present study, we identified Siah1 and Siah2 as novel binding partners of the PLCepsilon RA domain. Both Siah1 and Siah2 interacted with the RA2 domain of PLCepsilon, and the mutation of Lys-2186 of the PLCepsilon RA2 domain abolished this association. Moreover, Siah induced the ubiquitination and degradation of PLCepsilon upon epidermal growth factor (EGF) stimulation, and Siah proteins were phosphorylated on multiple tyrosine residues via an Src-dependent pathway upon EGF treatment. The Src inhibitor abolished the EGF-dependent ubiquitination of PLCepsilon, and the Siah1 phosphorylation-deficient mutant could not increase the EGF-dependent ubiquitination and degradation of PLCepsilon. The EGF-dependent degradation of PLCepsilon was blocked in mouse embryonic fibroblast (MEF) cells derived from Siah1a/Siah2 double knockout mice, and the extrinsic expression of wild-type Siah1 restored the degradation of PLCepsilon, whereas the phosphorylation-deficient mutant did not. Siah1 expression abolished PLCepsilon-dependent potentiation of EGF-dependent cell growth. In addition, the expression of wild-type Siah1 in Siah1a/Siah2-double knockout MEF cells inhibited EGF-dependent cell growth, and this inhibition was abolished by PLCepsilon knockdown. Our results suggest that the Siah-dependent degradation of PLCepsilon plays a role in the regulation of growth factor-dependent cell growth.
Highlights
phospholipase C (PLC)⑀ plays a role in the interplay between PLC and small GTPases
The immunocomplexes two-hybrid analysis revealed that the RA domain of PLC⑀ interwere subjected to SDS-PAGE and autoradiography
Siah1 and amounts of immunoprecipitated Siah proteins were measured Siah2 were identified as novel PLC⑀ RA domain-binding proby immunoblotting and used for the quantitation of relative teins
Summary
PLC⑀ plays a role in the interplay between PLC and small GTPases. Various G-proteins directly activate PLC⑀. EGF treatment was found to induce an association between the PLC⑀ RA domain and activated Ras, and this resulted in the recruitment of PLC⑀ into the plasma membrane for activation [8]. Several physiological studies have indicated that PLC⑀ is involved in development and cell growth. Ablation of PLC⑀ activity in mice led to reduced carcinogen-induced skin tumor formation, providing evidence that PLC⑀ plays a positive role in tumor cell growth [12]. Siah proteins are homologues of Drosophila SINA, which is a ring-finger protein involved in R7 cell development in the eye medium; MEF, mouse embryonic fibroblast; siRNA, small interfering RNA; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase. Siah proteins are RING finger proteins with E3 ligase activity and have been implicated in the ubiquitination and proteasomedependent degradation of various substrate molecules. Siah proteins are quite diverse and include transcriptional regulators [17,18,19], membrane receptors [20, 21], a microtubule-associated motor protein [22], and other pro-
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