Mouse class III myosins: kinase activity and phosphorylation sites

Selective autophagy of damaged mitochondria, protein aggregates, and other cargoes is essential for health. Cargo initiates phagophore biogenesis, which entails the conjugation of LC3 to phosphatidylethanolamine. Current models suggest that clustered ubiquitin chains on a cargo trigger a cascade from autophagic cargo receptors through the core complexes ULK1 and class III phosphatidylinositol 3-kinase complex I, WIPI2, and the ATG7, ATG3, and ATG12ATG5-ATG16L1 machinery of LC3 lipidation. This was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required all components, but not ULK1 kinase activity. However, OPTN bypassed the ULK1 requirement. Thus, cargo-dependent stimulation of LC3 lipidation is common to multiple autophagic cargo receptors, yet the details of core complex engagement vary between the different receptors.

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Atypical, Non-V600 BRAF Mutations as a Potential Mechanism of Resistance to EGFR Inhibition in Metastatic Colorectal Cancer

JCO Precision Oncology ◽

10.1200/po.19.00102 ◽

2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Benny Johnson ◽

Jonathan M. Loree ◽

Alexandre A. Jacome ◽

Shehara Mendis ◽

Muddassir Syed ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Stable Disease ◽

Kinase Activity ◽

Class Ii ◽

Activity Level ◽

Class Iii ◽

Braf Mutations ◽

Egfr Inhibition ◽

Mechanism Of Resistance

PURPOSE Atypical, non-V600 BRAF ( aBRAF) mutations represent a rare molecular subtype of metastatic colorectal cancer (mCRC). Preclinical data are used to categorize aBRAF mutations into class II (intermediate to high levels of kinase activity, RAS independent) and III (low kinase activity level, RAS dependent). The clinical impact of these mutations on anti-EGFR treatment efficacy is unknown. PATIENTS AND METHODS Data from 2,084 patients with mCRC at a single institution and from an external cohort of 5,257 circulating tumor DNA (ctDNA) samples were retrospectively analyzed. Overall survival (OS) was calculated using Kaplan-Meier and log-rank tests. Statistical tests were two-sided. RESULTS BRAF mutations were harbored by 257 patients, including 36 with aBRAF mutations: 22 class III, 10 class II, four unclassified. For patients with aBRAF mCRC, median OS was 36.1 months, without a difference between classes, and median OS was 21.0 months for patients with BRAFV600E mCRC. In contrast to right-sided predominance of tumors with BRAFV600E mutation, 53% of patients with aBRAF mCRC had left-sided primary tumors. Concurrent RAS mutations were noted in 33% of patients with aBRAF mCRC, and 67% of patients had microsatellite stable disease. Among patients with aBRAF RAS wild-type mCRC who received anti-EGFR antibodies (monotherapy, n = 1; combination therapy, n = 10), no responses to anti-EGFR therapy were reported, and six patients (four with class III aBRAF mutations, one with class II, and one unclassified) achieved stable disease as best response. Median time receiving therapy was 4 months (range, 1 to 16). In the ctDNA cohort, there was an increased prevalence of aBRAF mutations and subclonal aBRAF mutations ( P < .001 for both) among predicted anti-EGFR exposed compared with nonexposed patients. CONCLUSION Efficacy of anti-EGFR therapy is limited in class II and III aBRAF mCRC. Detection of aBRAF mutations in ctDNA after EGFR inhibition may represent a novel mechanism of resistance.

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Regulation of Jak2 Function by Phosphorylation of Tyr317 and Tyr637 during Cytokine Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00278-09 ◽

2009 ◽

Vol 29 (12) ◽

pp. 3367-3378 ◽

Cited By ~ 23

Author(s):

Scott A. Robertson ◽

Rositsa I. Koleva ◽

Lawrence S. Argetsinger ◽

Christin Carter-Su ◽

Jarrod A. Marto ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Feedback Inhibition ◽

Dominant Role ◽

Kinase Domain ◽

Cytokine Signaling ◽

Tyrosine Kinase Domain ◽

Phosphorylation Sites ◽

Cytokine Stimulation

ABSTRACT Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.

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Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

FEBS Letters ◽

10.1016/j.febslet.2012.04.046 ◽

2012 ◽

Vol 586 (11) ◽

pp. 1584-1591 ◽

Cited By ~ 63

Author(s):

Hilde Abrahamsen ◽

Harald Stenmark ◽

Harald W. Platta

Keyword(s):

Tumor Suppression ◽

Kinase Activity ◽

Beclin 1 ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Binding Partners ◽

Phosphatidylinositol 3

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Structural differences between repressed and derepressed forms of p60c-src

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2648-2656.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2648-2656

Author(s):

A MacAuley ◽

J A Cooper

Keyword(s):

Kinase Activity ◽

Kinase Domain ◽

Terminal Region ◽

Phosphorylation Sites ◽

Carboxy Terminus ◽

Kinase Activation ◽

Amino Terminal ◽

Structural Differences ◽

Carboxy Terminal ◽

Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

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Raf-1 activation disrupts its binding to keratins during cell stress

The Journal of Cell Biology ◽

10.1083/jcb.200402051 ◽

2004 ◽

Vol 166 (4) ◽

pp. 479-485 ◽

Cited By ~ 39

Author(s):

Nam-On Ku ◽

Haian Fu ◽

M. Bishr Omary

Keyword(s):

Cell Signaling ◽

Kinase Activity ◽

Cultured Cells ◽

Mutational Analysis ◽

Cell Stress ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Raf Kinase ◽

Toxin Exposure ◽

Keratin 14

Keratins 8 and 18 (K8/18) heteropolymers may regulate cell signaling via the known K18 association with 14-3-3 proteins and 14-3-3 association with Raf-1 kinase. We characterized Raf–keratin–14-3-3 associations and show that Raf associates directly with K8, independent of Raf kinase activity or Ras–Raf interaction, and that K18 is a Raf physiologic substrate. Raf activation during oxidative and toxin exposure in cultured cells and animals disrupt keratin–Raf association in a phosphorylation-dependent manner. Mutational analysis showed that 14-3-3 residues that are essential for Raf binding also regulate 14-3-3–keratin association. Similarly, Raf phosphorylation sites that are important for binding to 14-3-3 are also essential for Raf binding to K8/18. Therefore, keratins may modulate some aspects of Raf signaling under basal conditions via sequestration by K8, akin to Raf–14-3-3 binding. Keratin-bound Raf kinase is released upon Raf hyperphosphorylation and activation during oxidative and other stresses.

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PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817078116 ◽

2019 ◽

Vol 116 (5) ◽

pp. 1613-1620 ◽

Cited By ~ 12

Author(s):

Gonen Memisoglu ◽

Vinay V. Eapen ◽

Ying Yang ◽

Daniel J. Klionsky ◽

James E. Haber

Keyword(s):

Kinase Activity ◽

Budding Yeast ◽

Nutrient Starvation ◽

Phosphorylation Sites ◽

Phagophore Assembly Site ◽

Assembly Site

Macroautophagy is orchestrated by the Atg1-Atg13 complex in budding yeast. Under nutrient-rich conditions, Atg13 is maintained in a hyperphosphorylated state by the TORC1 kinase. After nutrient starvation, Atg13 is dephosphorylated, triggering Atg1 kinase activity and macroautophagy induction. The phosphatases that dephosphorylate Atg13 remain uncharacterized. Here, we show that two redundant PP2C phosphatases, Ptc2 and Ptc3, regulate macroautophagy by dephosphorylating Atg13 and Atg1. In the absence of these phosphatases, starvation-induced macroautophagy and the cytoplasm-to-vacuole targeting pathway are inhibited, and the recruitment of the essential autophagy machinery to the phagophore assembly site is impaired. Expressing a genomic ATG13-8SA allele lacking key TORC1 phosphorylation sites partially bypasses the macroautophagy defect in ptc2Δ ptc3Δ strains. Moreover, Ptc2 and Ptc3 interact with the Atg1-Atg13 complex. Taken together, these results suggest that PP2C-type phosphatases promote macroautophagy by regulating the Atg1 complex.

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Prevalence of class I–III BRAF mutations among 114,662 cancer patients in a large genomic database

Experimental Biology and Medicine ◽

10.1177/1535370220959657 ◽

2020 ◽

pp. 153537022095965

Author(s):

Jeff Owsley ◽

Matthew K Stein ◽

Jason Porter ◽

Gino K In ◽

Mohamed Salem ◽

...

Keyword(s):

Braf Mutation ◽

Kinase Activity ◽

Human Cancer ◽

Class Ii ◽

Class I ◽

Clinical Activity ◽

Class Iii ◽

Braf Mutations ◽

Comprehensive Genomic Profiling ◽

Relative Prevalence

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer. Impact statement These data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.

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Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation

International Journal of Molecular Sciences ◽

10.3390/ijms20246307 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6307 ◽

Cited By ~ 1

Author(s):

Aura E. Ionescu ◽

Mihaela Mentel ◽

Cristian V.A. Munteanu ◽

Livia E. Sima ◽

Eliza C. Martin ◽

...

Keyword(s):

Kinase Activity ◽

Src Kinase ◽

Protein Tyrosine Phosphatases ◽

Phosphorylation Sites ◽

Src Tyrosine Kinase ◽

Eyes Absent ◽

Tyrosine Residues ◽

Proliferative Effect ◽

Phosphorylation Pattern

Eyes absent (EYA) are non-thiol-based protein tyrosine phosphatases (PTPs) that also have transcriptional co-activator functions. Their PTP activity is involved in various pathologies. Recently, we demonstrated that Src tyrosine kinase phosphorylates human EYA3 by controlling its subcellular localization. We also found EYA3′s ability to autodephosphorylate, while raising the question if the two opposing processes could be involved in maintaining a physiologically adequate level of phosphorylation. Using native and bottom-up mass spectrometry, we performed detailed mapping and characterization of human EYA3 Src-phosphorylation sites. Thirteen tyrosine residues with different phosphorylation and autodephosphorylation kinetics were detected. Among these, Y77, 96, 237, and 508 displayed an increased resistance to autodephosphorylation. Y77 and Y96 were found to have the highest impact on the overall EYA3 phosphorylation. Using cell cycle analysis, we showed that Y77, Y96, and Y237 are involved in HEK293T proliferation. Mutation of the three tyrosine residues abolished the pro-proliferative effect of EYA3 overexpression. We have also identified a Src-induced phosphorylation pattern of EYA3 in these cells. These findings suggest that EYA3′s tyrosine phosphorylation sites are non-equivalent with their phosphorylation levels being under the control of Src-kinase activity and of EYA3′s autodephosphorylation.

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