Biology subjects

Kundinger, S. R.

Publications and source records attributed to Kundinger, S. R..

4 recordsLinked to original sources

bioRxiv2021

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Post-translational modifications (PTMs) within splicing factor RNA-binding proteins (RBPs), such as phosphorylation, regulate several critical steps in RNA metabolism including spliceosome assembly, alternative splicing and mRNA export. Notably, the arginine-/serine-rich (RS) domains in SR proteins are densely modified by phosphorylation compared with the remainder of the proteome. Previously, we showed that dephosphorylation of SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich to the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. Phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high molecular weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine-/arginine protein kinase 2 (SRPK2) in vitro prevented high molecular weight SRSF2 species formation. Furthermore, we pharmacologically inhibited SRPKs in mammalian cells and observed increased cytoplasmic granules as well as the formation of cytoplasmic SRSF2 tubular structures that associate with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

biochemistry↗

bioRxiv2020

Global quantitative analysis of the human brain proteome and phosphoproteome in Alzheimer's disease

Alzheimers disease (AD) is characterized by an early, asymptomatic phase (AsymAD) in which individuals exhibit amyloid-beta (A{beta}) plaque accumulation in the absence of clinically detectable cognitive decline. Here we report an unbiased multiplex quantitative proteomic and phosphoproteomic analysis using tandem mass tag (TMT) isobaric labeling of human post-mortem cortex (n=27) across pathology-free controls, AsymAD and symptomatic AD individuals. With off-line high-pH fractionation and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on an Orbitrap Lumos mass spectrometer, we identified 11,378 protein groups across three TMT 11-plex batches. Immobilized metal affinity chromatography (IMAC) was used to enrich for phosphopeptides from the same TMT-labeled cases and 51,736 phosphopeptides were identified. Of these, 48,992 were quantified by TMT reporter ions representing 33,652 unique phosphosites. Two reference standards in each TMT 11-plex were included to assess intra- and inter-batch variance at the protein and peptide level. This comprehensive human brain proteome and phosphoproteome dataset will serve as a valuable resource for the identification of biochemical, cellular and signaling pathways altered during AD progression.

neuroscience↗

bioRxiv2019

Middle-down proteomics reveals dense sites of methylation and phosphorylation in arginine-rich RNA-binding proteins

Arginine (Arg)-rich RNA-binding proteins play an integral role in RNA metabolism. Post-translational modifications (PTMs) within Arg-rich domains, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we report a middle-down proteomic approach coupled with electron transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of HEK293 cells. Remarkably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with di-methylation and phosphorylation favoring RSRS motifs. Although they favor a common motif, analysis of combinatorial PTMs within RSRS motifs indicate that phosphorylation and methylation do not often co-occur, suggesting they may functionally oppose one another. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome, and a possible unexplored regulator of RNA metabolism. These data also serve as a resource to facilitate future mechanistic studies of the role of PTMs in RNA-binding protein structure and function.\n\nBriefsMiddle-down proteomics reveals arginine-rich RNA-binding proteins contain many sites of methylation and phosphorylation.

biochemistry↗

bioRxiv2018

Kif17 phosphorylation regulates its ciliary localization and photoreceptor outer segment turnover

BackgroundKIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown.\n\nResultsUsing transgenic expression in both mammalian cells and zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization to cilia. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding.\n\nConclusionsTaken together, our data support a model in which phosphorylation of KIF17 promotes its ciliary localization. In cone photoreceptor outer segments, this promotes disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.

cell biology↗