biostudies-literatureNishiyama KTakeda Science Foundation to Proteo-Science CenterJSPS KAKENHISPS KAKENHIJapan Foundation for Applied Enzymology, Research Grant of the Princess Takamatsu Cancer Research Fund, The Research Foundation for Pharmaceutical Sciencese202101095https://www.ebi.ac.uk/biostudies/studies/S-EPMC8321701biostudies-literatureUnknown4(9)Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) phosphorylation drives HER2-positive breast cancer cell proliferation. Enforced activation of phosphatases for those receptors could be a therapeutic option for HER2-positive breast cancers. Here, we report that degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Using human protein arrays produced in a wheat cell-free protein synthesis system, RhoB-GTP, and protein tyrosine phosphatase receptor type H (PTPRH) were identified as interacting proteins of connector enhancer of kinase suppressor of Ras1 (CNKSR1). Mechanistically, constitutive degradation of RhoB, which is mediated by the CUL3Life science allianceCNKSR1 serves as a scaffold to activate an EGFR phosphatase via exclusive interaction with RhoB-GTP.PMC832170119K1803019K1802921H02763Higashiyama SNakayama JTakada YKiyoi TNakagita TNishiyama KMurakami AKamei YChosei MMaekawa MTakeda HfalseCNKSR1 serves as a scaffold to activate an EGFR phosphatase via exclusive interaction with RhoB-GTP.Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) phosphorylation drives HER2-positive breast cancer cell proliferation. Enforced activation of phosphatases for those receptors could be a therapeutic option for HER2-positive breast cancers. Here, we report that degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Using human protein arrays produced in a wheat cell-free protein synthesis system, RhoB-GTP, and protein tyrosine phosphatase receptor type H (PTPRH) were identified as interacting proteins of connector enhancer of kinase suppressor of Ras1 (CNKSR1). Mechanistically, constitutive degradation of RhoB, which is mediated by the CUL32021-01-01T00:00:00Z2021 Sep2026-05-08T13:04:32.231Z2022-02-11T04:14:29.304ZS-EPMC83217013418793410.26508/lsa.202101095