During the fiscal year, we accomplished the following: 1. Solved the NMR structure of calmodulin bound to estrogen receptor- (ER). Prior work from our laboratory identified and characterized the functional interactions of calmodulin with non-classic calmodulin target proteins, including IRS-1 (insulin receptor substrate-1), IRS-2, IQGAP1, phosphatidylinositol 3-kinase (PI3K), p21Cip, androgen receptor, epidermal growth factor receptor (EGFR, also termed HER1) and ER. Calmodulin binds directly to ER (which stimulates tumorigenesis in ER(+) breast carcinoma) in a Ca2+-dependent manner. We documented that calmodulin binding both promotes the stability of ER by sequestering it away from the ubiquitin-proteasome pathway and is necessary for estrogen-stimulated transcriptional activity of ER. We previously identified the calmodulin-binding domain on ER. In collaboration with Jim Ames, we solved the NMR structures of the full-length calmodulin bound to two ER peptides. The two lobes of calmodulin bind to the same site on two separate ER molecules. Calmodulin binds two molecules of ER in a 1:2 complex and stabilizes ER dimerization. Full-length calmodulin increased the ability of estrogen to enhance transcriptional activation by ER of endogenous estrogen responsive genes. By contrast, expression of the individual lobes of calmodulin, either the N- or C-lobe, abrogated estrogen-stimulated transcription. These data suggest that small molecules that selectively disrupt the interaction of calmodulin with ER may be useful in the therapy of breast carcinoma. 2. Showed that knockdown of IQGAP1 in endothelial cells by siRNA abolished oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC)-induced enlargement of VE-cadherin-positive adherens junctions, suppressed peripheral accumulation of actin polymerization regulators and attenuated remodeling of the peripheral actin cytoskeleton. Inhibition of OxPAPC-induced barrier enhancement by IQGAP1 knockdown was due to suppressed Rac1 and Cdc42 activation. Expression of an IQGAP1 truncated mutant showed that the GTPase regulatory domain (GRD) of IQGAP1 was essential for the OxPAPC-induced membrane localization of cortactin, adherens junction proteins VE-cadherin and p120-catenin as well as for EC permeability response. IQGAP1knockdown attenuated the protective effect of OxPAPC against thrombin-induced cell contraction, cell junction disruption and EC permeability. These results demonstrate for the first time the role of IQGAP1 as a critical transducer of OxPAPC-induced Rac1/Cdc42 signaling to the actin cytoskeleton and adherens junctions which promotes cortical cytoskeletal remodeling and EC barrier protective effects of oxidized phospholipids. 3. Established a mechanism forphosphatidylinositol-3,4,5-trisphosphate PtdIns(3,4,5)P3generation by scaffolded phosphoinositide kinases. In this pathway, class I phosphatidylinositol-3-OH kinase(PI(3)K) is assembled by IQGAP1 with PI(4)KIII and PIPKI, which sequentially generate PtdIns(3,4,5)P3from phosphatidylinositol. By scaffolding these kinases into functional proximity, the PtdIns(4,5)P2generated isselectively used by PI(3)K for PtdIns(3,4,5)P3generation, which then signals to PDK1 and Akt that are also in the complex. Moreover, multiple receptor types stimulate the assembly of this IQGAP1-PI(3)K signalling complex. Blockade of IQGAP1 interaction with PIPKI or PI(3)K inhibited PtdIns(3,4,5)P3generation and signalling, and selectively diminished cancer cell survival, revealing a target for cancer chemotherapy. 4. Showed that IQGAP1 associates with both the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), and influences insulin action. Insulin binds to IR and induces tyrosine phosphorylation of the receptor and IRS-1, leading to activation of the PKB/Akt and MAPK/ERK pathways.In vitroanalysis with pure proteins revealed that the IQ region of IQGAP1 binds directly to the intracellular domain of IR. Similarly, the phosphotyrosine-binding domain of IRS-1 mediates a direct interaction with the C-terminal tail of IQGAP1. Consistent with these observations, both IR and IRS-1 co-immunoprecipitated with IQGAP1 from cells. Investigation of the functional effects of the interactions revealed that in the absence of IQGAP1, insulin-stimulated phosphorylation of Akt and ERK, as well as the association of phosphatidylinositol 3-kinase with IRS-1, were significantly decreased. Importantly, loss of IQGAP1 results in impaired insulin signaling and glucose homeostasisin vivo.Collectively, these data reveal that IQGAP1 is a scaffold for IR and IRS-1 and implicate IQGAP1 as a participant in insulin signaling.
Showing the most recent 10 out of 34 publications
