IQGAP1 in tumorigenesis

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Abstract

During the fiscal year we accomplished the following: 1. During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. Separately, calmodulin is a Ca2+-dependent protein that modulates the activity of target proteins and regulates several signaling cascades; however, its potential role in the Hippo pathway has not been identified. We demonstrated that calmodulin promotes Hippo signaling. We showed that purified YAP and LATS1 bind directly to calmodulin and form a Ca2+-dependent ternary complex in vitro. Importantly, Ca2+/calmodulin directly stimulated the activity of LATS1 kinase. In cultured mammalian cells, we demonstrated that endogenous YAP and LATS1 coimmunoprecipitate with endogenous calmodulin. In cells with activated Hippo signaling, we show that calmodulin antagonism significantly (i) decreases YAP phosphorylation, (ii) increases expression of two Hippo target genes (connective tissue growth factor CTGF and cysteine-rich angiogenic inducer 61 CYR61) that regulate cell proliferation and tumor progression, and (iii) enhances the interaction of YAP with its major transcription factor, thereby facilitating transcription of target genes. Collectively, our data demonstrate that calmodulin activates the Hippo kinase cascade and inhibits YAP activity via a direct interaction with LATS1 and YAP, thereby uncovering previously unidentified crosstalk between the Ca2+/calmodulin and Hippo signaling pathways. 2. Melanoma is the most aggressive skin malignancy with increasing incidence globally. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. We demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of -catenin, a key transcription factor in the Wnt pathway. The amount of -catenin in cells was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of -catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with -catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and -catenin interact as potential new components of the Wnt signaling pathway.