Coordinated membrane and actin remodeling are integral to a number of cellular functions, including (i) cell movement, (ii) endocytosis and exocytosis, and (iii) mitosis and are central to pathological processes such as tumor cell invasion and metastasis. A number of signaling molecules that control either or both membrane and actin remodeling include phosphoinositides, Arf family GTP-binding proteins and Rho family GTP-binding proteins. The main objective of the work in our laboratory is to elucidate the mechanisms that regulate signals mediated by Arf family proteins. The work has led to the identification of a family of Arf GTPase-activating proteins, the AZAPs, that may integrate at least four signaling pathways, providing coordinated responses in membranes and actin necessary for complex cellular behaviors. The AZAPs are comprised of four subfamilies: ASAP, ACAP, AGAP and ARAP. Three AZAP family members have been directly implicated in carcinogenesis. Studies with two general goals are being conducted. One emphasis of the laboratory is to examine specific molecular mechanisms by which Arf GAPs interact with Arf and regulate Arf function. In these studies, we will determine (i) the interfaces between Arfs and GAPs; (ii) the interfaces between Arfs and coat proteins; (iii) the catalytic mechanism leading to GTP hydrolysis; (iv) mechanisms regulating GAP activity; and (v) functional interactions among Arfs, GAPs and Arf effectors including coat proteins. Studies to date have led us to propose (i) a new paradigm for the molecular mechanism by which Arf regulates cargo sorting and membrane trafficking, and (ii) an effector function for Arf GAPs of the AZAP family. Ongoing studies include testing these hypotheses and examining Arf GAPs as targets of signaling through tyrosine kinases and phosphoinositides. A second emphasis of the laboratory is an examination of the specific cellular sites of action of AZAP family members.
This aim i s closely related to the first. Using several strategies we have been determining the site of action for representative Arf GAPs. We have found specific membrane trafficking compartments regulated by some Arf GAPs. For instance, AGAP1 regulates AP-3 endosomes and ARAP1 regulates EGFR endocytic traffic. We have also found that specific cytoskeletal structures are regulated by other Arf GAPs. For instance, ASAP1 regulates focal adhesions, circular dorsal ruffles and invadopodia. We are currently examining the specific mechanisms that maintain and regulate Arf GAPs at specific sites, and exploring hypotheses about how the membrane trafficking sites may be related to the cytoskeletal sites. The hypotheses we are currently testing are (i) that Arf GAPs are in the Rho family signaling pathway with some Arf GAPs functioning as Rho effectors and (ii) Arf GAPs are targets of Src that are of crucial importance for the formation of podosomes and their pathologic analogs invadopia.iii. that Arf GAPs regulate proliferative and survival signaling by control of Ras signaling and transcriptional reprogramming. Z01 BC 07365-03 LBC to LCO
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