Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates

Chou, Michael F.; Prisic, Sladjana; Lubner, Joshua M.; Church, George M.; Husson, Robert N.; Schwartz, Daniel

doi:10.1371/journal.pone.0052747

Cited by 27 publications

(44 citation statements)

References 48 publications

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“…The clearly defined pattern of phosphorylation observed in the context of peptides harboring a fixed S/T phosphoacceptor sequence suggests that Ssp2 KAD /Smk1 is predominantly an S/T kinase and that Y kinase activity on random peptide substrates is undetectable. We also compared the sequences surrounding the phosphorylated peptides identified in the bacterial phosphoproteomic analysis by using a program that normalizes amino acid occurrence in the set of pT-and pS-containing phosphopeptides to the relative abundance in the proteome (pLogo) (23,24) (Fig. 6C; Table S1).…”

Section: Resultsmentioning

confidence: 99%

Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase

Tio

Omerza

Phillips

et al. 2017

Molecular and Cellular Biology

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Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in Saccharomyces cerevisiae that couples spore morphogenesis to the completion of chromosome segregation. Similar to other MAPKs, Smk1 is controlled by phosphorylation of a threonine (T) and a tyrosine (Y) in its activation loop. However, it is not activated by a dual-specificity MAPK kinase. Instead, T207 in Smk1's activation loop is phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase (Cak1), and Y209 is autophosphorylated in an intramolecular reaction that requires the meiosis-specific protein Ssp2. In this study, we show that Smk1 is catalytically inert unless it is bound by Ssp2. While Ssp2 binding activates Smk1 by a mechanism that is independent of activation loop phosphorylation, binding also triggers autophosphorylation of Y209 in Smk1, which, along with Cak1-mediated phosphorylation of T207, further activates the kinase. Autophosphorylation of Smk1 on Y209 also appears to modify the specificity of the MAPK by suppressing Y kinase and enhancing S/T kinase activity. We also found that the phosphoconsensus motif preference of Ssp2/Smk1 is more extensive than that of other characterized MAPKs. This study therefore defines a novel mechanism of MAPK activation requiring binding of an activator and also shows that MAPKs can be diversified to recognize unique phosphorylation motifs.

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Section: Resultsmentioning

confidence: 99%

Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase

Tio

Omerza

Phillips

et al. 2017

Molecular and Cellular Biology

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“…23 His 6 /V5-tagged ATG16L1 expressed in E. coli was purified using Ni-NTA beads followed by CSNK2 treatment, and then analyzed for its association with purified ATG12-ATG5 conjugate. The results indicated that CSNK2 phosphorylation of ATG16L1 promoted its association with the ATG12-ATG5 protein complex, which was abolished by dephosphorylation of ATG16L1 by lPP (Fig.…”

Section: Csnk2 Was Responsible For Atg16l1 Phosphorylation In H/rtreamentioning

confidence: 99%

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

Song¹,

Wang³

et al. 2015

Autophagy

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(2015) ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation, Autophagy, 11:8, 1308-1325, DOI: 10.1080/15548627.2015 Keywords: ATG16L1, autophagy, cardiomyocyte, casein kinase 2, protein phosphatase 1 Abbreviations: ACTB, actinb; AMPK, AMP activated protein kinase; ATG, autophagy-related; BCL2, B-cell CLL/lymphoma 2; BECN1/Beclin 1, autophagy related; CD, Crohn disease; CSNK2, casein kinase 2; GFP, green fluorescent protein; GNB2L1/ RACK1, guanine nucleotide binding protein (G protein)bpolypeptide 2-like 1; GST, glutathione S-transferase; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 b; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin complex 1; NRVCs, neonatal rat ventricular cardiomyocytes; OA, okadaic acid; PE, phosphatidylethanolamine; PPP1, protein phosphatase 1; PPP2, protein phosphatase 2; PVDF, polyvinylidenedifluoride; SNP, single nucleotide polymorphism; SUPT20H/p38IP, suppressor of Ty 20 homolog (S. cerevisiae); TBB, 4,5,6,7-tetrabromobenzotriazole; lPP, l protein phosphatase.Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/ reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autoph...

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“…To bypass endogenous kinase activity, motifs were also revealed by mass spectrometry for recombinant serine/threonine kinases PKA and CK2 expressed in bacteria (Chou et al, 2012). Utilizing experimentally derived motif data, a variety of computational approaches for scoring linear kinase motifs in the proteome to predict putative substrate sites have been developed (Miller et al, 2008;Xue et al, 2008;Hu et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate

et al. 2017

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Systematic assessment of tyrosine kinase-substrate relationships is fundamental to a better understanding of cellular signaling and its profound alterations in human diseases such as cancer. In human cells, complex signaling networks, feedback loops, conditional activity and intra-kinase redundancy combine to confound systematic attempts to address this topic. We leveraged the model organism S. cerevisiae to individually express human non-receptor tyrosine kinases (NRTKs) and exploited the full yeast proteome as an in vivo model substrate. For 16 NRTKs, we recorded 3,279 kinase-substrate relationships involving 1,351 yeast pY-sites. From this data we generated a set of new linear kinase motifs and assigned ~1,300 known human pY-sites to specific NRTKs. Furthermore, experimentally defined pY-sites for each individual kinase were shown to cluster within the yeast interactome network irrespective of linear motif information. We therefore applied a network inference approach to predict kinase-substrate relationships for more than 3,500 human proteins, marking a substantial step forward in our understanding of kinase biology.. Corwin et al., revised3 7/25/2017 3

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Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates

Cited by 27 publications

References 48 publications

Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase

Ssp2 Binding Activates the Smk1 Mitogen-Activated Protein Kinase

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate

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