Cellular processing of RNF13, an E3 in the ubiquitin ligase pathway (2011)

Undergraduates: Taufiq Salahuddin, none none none

Faculty Advisor: Ann Erickson
Department: Chemistry

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RING finger protein 13 (RNF13) is a ubiquitously expressed, highly regulated ubiquitin ligase (E3) anchored in endosome membranes whose substrates are currently unknown. Regulatory proteases clip the protein to generate soluble and membrane-bound isoforms containing the catalytically active RING domain. RNF13 is targeted to the nuclear membrane upon activation of protein kinase C, exposing the RING domain to the nucleoplasm. A mutagenesis study was undertaken to disrupt RNF13’s regulatory processing, nuclear localization, and physiological function. The ectopic expression of RNF13 with a deletion of positively charged residues composing a putative nuclear localization signal (NLS) displays differences in cell adhesion properties, implicating the protein in cell-cell matrix interactions. This putative NLS deletion also reduces the observed MW of the protein, presumably by disrupting normal post-translational modifications. A point mutation in the RING domain changes the stability of the protein as observed by SDS-PAGE, presumably by interfering with E2 binding and resulting in loss of activity, while maintaining normal cellular localization observed by immunofluorescence microscopy (IF). Mutations of positively charged residues on the N-terminal juxtamembrane region (potential serine protease sites) result in changes in proteolytic processing similar to that observed with the inhibition of gamma-secretase.