Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system

Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation. © 2016 Nature America, Inc

Short Linear Motifs recognized by SH2, SH3 and Ser/Thr Kinase domains are conserved in disordered protein regions

<p>Abstract</p> <p>Background</p> <p>Protein interactions are essential for most cellular functions. Interactions mediated by domains that appear in a large number of proteins are of particular interest since they are expected to have an impact on diversities of cellular processes such as signal transduction and immune response. Many well represented domains recognize and bind to primary sequences less than 10 amino acids in length called Short Linear Motifs (SLiMs).</p> <p>Results</p> <p>In this study, we systematically studied the evolutionary conservation of SLiMs recognized by SH2, SH3 and Ser/Thr Kinase domains in both ordered and disordered protein regions. Disordered protein regions are protein sequences that lack a fixed three-dimensional structure under putatively native conditions. We find that, in all these domains examined, SLiMs are more conserved in disordered regions. This trend is more evident in those protein functional groups that are frequently reported to interact with specific domains.</p> <p>Conclusion</p> <p>The correlation between SLiM conservation with disorder prediction demonstrates that functional SLiMs recognized by each domain occur more often in disordered as compared to structured regions of proteins.</p

The structure of an NDR/LATS kinase – mob complex reveals a novel kinase-coactivator system and substrate docking mechanism.

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. "Hippo" pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with "Mob" coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1-Mob2, to our knowledge the first of an NDR/LATS kinase-Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1's regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides

Investigating how ubiquitin-mediated proteolysis of AURKA contributes to its activity in the cell cycle

Aurora kinase A (AURKA) is a major mitotic regulatory kinase required for mitotic entry, the formation of a bipolar mitotic spindle, and the completion of cytokinesis. In recent years AURKA has been identified as an upstream regulator for many interphase functions such as cilia disassembly and mitochondrial fragmentation. AURKA is overexpressed in many tumours and has a pivotal role in the acquisition of malignant cell phenotypes. Therefore, it is considered a highly attractive drug target for anti-cancer therapy. The activity of AURKA is regulated by phosphorylation at the active loop or the interaction with binding partners. TPX2 is a well-known binding partner of AURKA. It activates AURKA through stabilizing the T-loop and is required for targeting AURKA to the mitotic spindle. Phosphorylation and binding partners may act synergistically to induce hyperactivity of the kinase. Previous research from my lab has highlighted that AURKA is frequently co-expressed with TPX2 in human cancers and proposed AURKA/TPX2 complex as an oncogenic holoenzyme in a variety of cancers. AURKA protein is targeted for proteasome-mediated degradation by the FZR1 activated form of APC/C at the end of mitosis. This study focuses on characterisation of AURKA degrons, the contribution of APC/C-FZR1 in the timing of AURKA inactivation, identifying the physiological consequences of AURKA deregulation outside mitosis, and examining the role of Short Linear motifs (SLiMs) within AURKA N-terminal domain in regulating its stability and activity. I show that the previously known D-box-like motif (R371xxL374) within C-terminal is not a functional degron. I also reveal that the A-box motif may act as an atypical D-box that is sufficient to drive protein degradation. I use a new tool CRISPR/Cas9 FZR1 knockout cell line and a FRET-based biosensor for measuring AURKA activity to investigate directly whether AURKA inactivation is regulated simply by destruction. These, in combination with time-lapse imaging, show that inactivation of AURKA is identical in wild-type and FZR1KO cells, despit¬e the difference in protein levels between the two cell lines. I demonstrate that the timing of AURKA inactivation is regulated via the degradation of its activator TPX2 at mitotic exit. Moreover, the destruction of AURKA is required to regulate its interphase activity. I also identify that extra AURKA activity can have consequences on the morphology of the mitochondrial network outside of mitosis. My time-lapse imaging reveals that FZR1-restricted degradation of AURKA controls mitochondrial dynamics. This mechanism links the destruction machinery, through AURKA signaling to the mitochondrial dynamics of the cell. I further explore the role of the N-terminal domain in the regulation of AURKA activity through the detailed analysis of the potential SLiMs. I find that K23RVL has a role in mediating the autoinhibition of AURKA. I then investigate the hypothesis that calmodulin (CaM) protects AURKA from degradation through its binding to the A-box SLiM. I find that AURKA degradation is not affected by inhibition of Ca2+/CaM. In summary, this work sheds light not only on the molecular mechanisms of AURKA activity and stability but also on the physiological relevance outside mitosis, which is urgently needed in the field to understand the oncogenic activity of AURKA and to improve therapeutic applications of cancer patients.Cambridge Overseas Trust, Youssef Jameel Foundation and Cambridge Philosophical Society

Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates

Identification and characterization of chloroplast casein kinase II from Oryza sativa (rice)

Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolutio