Non-degradative Ubiquitination of Protein Kinases

Abstract

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

A regulatory interface on RIPK2 is required for XIAP binding and NOD signaling activity

“…Our work closes this gap and we confirm by two complementary, mass-spectrometry-based approaches in endogenous systems, that RIPK2 is ubiquitinated on multiple lysine residues. This is not unusual as many proteins become ubiquitinated on multiple sites during signaling [46,47]. Typically, there appears to be flexibility in the lysine residues that can be ubiquitinated and often, as was the case here, mutation of a single lysine has little impact on signaling.…”

Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling

“…and CD4 during infection (Ball et al, 2016). 24 hours post-infection, cells were lysed and lysates derived from HIV-1-and mock-infected cells were combined at equal protein concentrations and subjected to trypsin digestion, ubiquitin remnant immunoaffinity enrichment, and quantitative mass spectrometry analysis on an Orbitrap Elite mass spectrometry system (Xu et al, 2010).…”

“…In order to stabilize ubiquitinated substrates that may be rapidly degraded by the proteasome, all ubiquitination experiments were performed in both the presence and absence of a proteasome inhibitor, MG-132, provided at 10 μ M for 4 hours prior to harvesting. We have previously demonstrated that this treatment is sufficient to stabilize ubiquitinated peptides derived from canonical HIV-1 ubiquitination targets APOBEC3C and CD4 during infection (Ball et al, 2016). 24 hours post-infection, cells were lysed and lysates derived from HIV-1-and mock-infected cells were combined at equal protein concentrations and subjected to trypsin digestion, ubiquitin remnant immunoaffinity enrichment, and quantitative mass spectrometry analysis on an Orbitrap Elite mass spectrometry system (Xu et al, 2010).…”

“…We have previously demonstrated that differential ubiquitination of APOBEC3C and CD4 in HIV-1-infected cells could only be observed in the presence of proteasome inhibition (Ball et al, 2016). By combining ubiquitination profiles of proteasomeinhibited, HIV-1-infected cells with protein abundance profiles of HIV-1-infected cells in which the proteasome was not inhibited, we rapidly identified PP2A-B56, UNG, β catenin, and histone H1 isoforms as ubiquitination events that are destined for degradation by the proteasome.…”

Peptides meet ubiquitin: Simple interactions regulating complex cell signaling

“…Early research was focused on the role of ubiquitin as a molecular tag for protein degradation by the proteasome 26S . More recently, the non‐degradative roles of ubiquitin have emerged as major regulators of cell homeostasis, including DNA repair and endocytosis . Such activity is mediated by the non‐covalent interaction of ubiquitin‐modified proteins with diverse and divergent peptide motifs that are involved in the regulation of signal transduction networks.…”