Regulation of Yersinia Protein Kinase A (YpkA) Kinase Activity by Multisite Autophosphorylation and Identification of an N-terminal Substrate-binding Domain in YpkA

Pha, Khavong; Wright, Matthew; Barr, Tasha M.; Eigenheer, Richard A.; Navarro, Lorena

doi:10.1074/jbc.m114.601153

Cited by 8 publications

(14 citation statements)

References 43 publications

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“…This suggests that being able to bind actin either directly or indirectly serves as an important selection criterion. Navarro’s group has shown that YopO binds to and phosphorylates Gαq, the heterotrimeric G protein complex α subunit31. We did not detect either Gαq or its phosphorylated sequences in KISS.…”

Section: Discussioncontrasting

confidence: 79%

Mechanisms of Yersinia YopO kinase substrate specificity

Lee

Singaravelu

Wee

et al. 2017

Sci Rep

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Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO’s substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.

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Section: Discussioncontrasting

confidence: 79%

Mechanisms of Yersinia YopO kinase substrate specificity

Lee

Singaravelu

Wee

et al. 2017

Sci Rep

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“…Further analysis showed that mutation of all serine residues to alanine within amino acids 436-710 to alanine does not affect YpkA kinase activity in vitro or in cellulo. Moreover, mutation of serine residues to alanine within amino acids 1-150 or 150-400 results in decreased YpkA autophosphorylation in vitro, but has no effect on its kinase activity in cellulo; however, mutation of all serine residues to alanine within amino acids 1-400 results in an inactive YpkA kinase mutant [66] . Together, the results suggest that once translocated YpkA autophosphorylates on multiple serine residues within its N-terminus to activate its kinase domain.…”

Section: Kinase Activation Of Ypka and The Targeting Of Actin Regulatmentioning

confidence: 99%

“…Intriguingly though, multiple regulatory domains also overlap with the Sec/Trans domain of YpkA. To date, the chaperone binding domain (amino acids 20-77), the membrane localization domain (MLD; amino acids , and the substrate-binding domain (SBD; amino acids [40][41][42][43][44][45][46][47][48][49] all overlap with the Sec/Trans domain [65,66] . Salomon et al [67] also showed that residues located …”

Section: The Multi-domains Of Ypka/yopomentioning

confidence: 99%

“…The crystal structure of the Y. enterocolitica YopO-actin complex showed that actin binding allosterically positions the catalytic and activation loops of YpkA in an active conformation [69] . Actin binding induces YpkA autophosphorylation on Ser90 and Ser95 in vitro [59,66,68] . Interestingly, mutation of Ser90 and Ser95 to alanine on YpkA does not affect its kinase activity in cellulo whereas mutation of all serine residues on YpkA resulted in a catalytically inactive kinase [66] .…”

Section: Kinase Activation Of Ypka and The Targeting Of Actin Regulatmentioning

confidence: 99%

“…Actin binding induces YpkA autophosphorylation on Ser90 and Ser95 in vitro [59,66,68] . Interestingly, mutation of Ser90 and Ser95 to alanine on YpkA does not affect its kinase activity in cellulo whereas mutation of all serine residues on YpkA resulted in a catalytically inactive kinase [66] . Further analysis showed that mutation of all serine residues to alanine within amino acids 436-710 to alanine does not affect YpkA kinase activity in vitro or in cellulo.…”

Section: Kinase Activation Of Ypka and The Targeting Of Actin Regulatmentioning

confidence: 99%

See 2 more Smart Citations

Yersiniatype III effectors perturb host innate immune responses

Pha

Navarro

2016

WJBC

Self Cite

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The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type Ⅲ secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector proteins and their contribution to Yersinia pathogenesis.

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Structural insight into effector proteins of Gram‐negative bacterial pathogens that modulate the phosphoproteome of their host

2015

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Invading pathogens manipulate cellular process of the host cell to establish a safe replicative niche. To this end they secrete a spectrum of proteins called effectors that modify cellular environment through a variety of mechanisms. One of the most important mechanisms is the manipulation of cellular signaling through modifications of the cellular phosphoproteome. Phosphorylation/dephosphorylation plays a pivotal role in eukaryotic cell signaling, with~500 different kinases and~130 phosphatases in the human genome. Pathogens affect the phosphoproteome either directly through the action of bacterial effectors, and/or indirectly through downstream effects of host proteins modified by the effectors. Here we review the current knowledge of the structure, catalytic mechanism and function of bacterial effectors that modify directly the phosphorylation state of host proteins. These effectors belong to four enzyme classes: kinases, phosphatases, phospholyases and serine/threonine acetylases.

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Regulation of Yersinia Protein Kinase A (YpkA) Kinase Activity by Multisite Autophosphorylation and Identification of an N-terminal Substrate-binding Domain in YpkA

Cited by 8 publications

References 43 publications

Mechanisms of Yersinia YopO kinase substrate specificity

Mechanisms of Yersinia YopO kinase substrate specificity

Yersiniatype III effectors perturb host innate immune responses

Structural insight into effector proteins of Gram‐negative bacterial pathogens that modulate the phosphoproteome of their host

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