Seven‐In‐Absentia (SINA) Family E3 Ligases in Development and Growth

The RAS signal transduction cascade is a pivotal signaling pathway that controls numerous fundamental cellular processes, such as cell proliferation, differentiation, motility, and apoptosis. Oncogenic K‐RAS pathway activation is detected in approximately 30% of all human cancers. Seven‐IN‐Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway. Underscoring the importance of SINA is its high evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling hub and the downstream signaling “gatekeeper” in the RAS pathway, SIAHs are uniquely positioned to inhibit “undruggable” oncogenic K‐RAS activation that is prevalent in high‐grade metastatic cancer. Thus, it is important to delineate the enzymatic activity, molecular regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the elegant and powerful Drosophila photoreceptor cell development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, suggesting that SINA is an essential gene in development. All other sina mutant alleles exhibit stronger phenotypes than the previously published sina 2 and sina 3 alleles, providing us with an opportunity to study sina loss‐of‐function mutant phenotypes. Sequencing analysis of these newly identified mutant alleles reveals the critical roles of several immutatable amino acid residues essential for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we have generated a collection of transgenic fly lines that carry either wild‐type (WT) or dominant negative (DN) Dm SINA and human SIAH. The altered Dm SINA/hSIAH expression under the control of sev ‐, GMR ‐, and dpp ‐ and salivary gland ‐GAL4 drivers revealed the biological consequences of gain of function and/or loss of function of Dm SINA/hSIAH1/2 in transmitting RAS signaling in central nervous system (CNS) and peripheral nervous system (PNS) development. Immunofluorescent staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINA WT/DN /SIAH WT/DN in neurons resulted in dramatic changes in neuronal cell fate in the developing eye and notum. Furthermore, immunofluorescent staining of larval salivary glands revealed a critical role for SINA family E3 ligases in regulation of cell shape, focal adhesions, and cellular junctions. The results from these transgenic animals suggested that Drosophila SINA and human SIAH1/SIAH2 are evolutionarily conserved and functionally interchangeable. Through this developmental neurobiology and cellular biology‐based study, we aim to dissect the molecular action of SINA/SIAH1/2 in RAS signaling. Furthermore, we hope to translate these findings to demonstrate the anticancer efficacy of the next‐generation anti‐SIAH‐based anti‐K‐RAS strategy against high‐grade metastatic human cancer in the future. Support or Funding Information Extramural funding support provided by NIH NIGMS R01 GM069922Z‐05S1 and NCI R01 CA140550. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .