Conjugation of Complex Polyubiquitin Chains to WRNIP1

Abstract

Werner helicase interacting protein 1 (WRNIP1) is a ubiquitin-binding protein that undergoes extensive post-translational modification including ubiquitination, sumoylation, and phosphorylation. These post-translational modifications are expected to regulate the function of WRNIP1 in the DNA damage response. In this study, we use a denaturing tandem affinity purification technique along with mass spectrometry to show that, unlike most ubiquitin-binding proteins, WRNIP1 is polyubiquitinated. WRNIP1 polyubiquitination is reminiscent of the well-characterized phenomenon of the coupled monoubiquitination of ubiquitin-binding proteins in that this polyubiquitination is dependent on the presence of an intact ubiquitin-binding domain. The polyubiquitin chains conjugated to WRNIP1 are linked through lysines 11, 48, and 63. This study presents the first evidence for the conjugation of K11-K48-K63 polyubiquitin chains to a specific substrate in vivo. Polyubiquitination is likely to regulate WRNIP1's function in the DNA damage response, as UV radiation induces the hyperubiquitination of WRNIP1. Polyubiquitination with noncanonical intraubiquitin linkages may represent a unique mode of regulation of UBZ domain-containing proteins.

The discovery of a Werner helicase interacting protein (WHIP) association with the nuclear pore complex

“…43,44 The WHIP rim staining is not as prominent as the classic MAb 414 staining, but is detectable, as seen in a previous study of WHIP by Mano 38 and consistent with our earlier discovery of WHIP's observed separate pools of polyubiquitinated and sumoylated WHIP to exist in the cell. 43,44 The modifications of WHIP most likely regulate interaction, localization and function. These pools have consequences in the location and diverse functions of WHIP.…”

“…WHIP is highly modified; it has been found to be a target of phosphorylation, sumoylation and polyubiquitination. 32,43,44 These findings suggest that only the higher MW form is associated at the NE and the lower MW form is localized to the NP.…”

“…2A and CE Lane 1) at 80-82 kDa and slightly above 97 kDa. 32,43 A sample of isolated NP contains mostly a single band of lower MW ( Fig. 2A, Lane 2) with only a faint hint of a higher MW band.…”

“…33 WHIP may be a modulator for initiation or re-initiation events during DNA polymerase delta-mediated DNA synthesis and with an intrinsic ATPase activity that may function as a sensor of DNA damage or of arrested replication forks and regulates the extent of DNA synthesis. 36,43,44 Mutations of the yeast WHIP homologue, MGS1 (maintenance of genome stability 1), can enhance the accelerated aging process in budding yeast. 34,45,46 The present investigation was undertaken to substantiate the interaction of WHIP with the NPC, followed by visualization of WHIP at the NE.…”

Arginine Methylation Controls the Subcellular Localization and Functions of the Oncoprotein Splicing Factor SF2/ASF

“…We digested and processed the bands for analysis as described previously (2). We analyzed the tryptic peptides either by liquid chromatography (LC)-tandem MS (MS/MS) on a linear trap quadrupole mass spectrometer (Thermo) or by targeted fragmentation on a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometer (Applied Biosystems) (8). We analyzed the LC-MS/MS runs using the X!Tandem search engine (19) with the following parameters: variable modifications included mono-, di-, and trimethylation of Lys and mono-or dimethylation of Arg residues.…”