scholarly journals Structural basis for Rab8a GTPase recruitment of RILPL2 via LRRK2 phosphorylation of switch 2

2019 ◽  
Author(s):  
Dieter Waschbüsch ◽  
Elena Purlyte ◽  
Prosenjit Pal ◽  
Emma McGrath ◽  
Dario R. Alessi ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Effector Proteins ◽  
Structural Basis ◽  
Rab Gtpases ◽  
Post Translational Modification ◽  
Serine Threonine Kinase ◽  
Dynamic Regulation ◽  
Threonine Kinase ◽  
Membrane Protrusions ◽  
General Mode

AbstractRab8a GTPase is associated with the dynamic regulation of membrane protrusions in polarized cells. Rab8a is one of several Rab-family GTPases that are substrates of leucine-rich repeat kinase 2 (LRRK2), a serine/threonine kinase that is linked to inherited Parkinson’s disease. Rab8a is phosphorylated at T72 (pT72) in its switch 2 helix and the post-translational modification facilitates phospho-Rab8a (pRab8a) interactions with RILPL2, which subsequently regulates ciliogenesis. Here we report the crystal structure of pRab8a in complex with the phospho-Rab binding domain of RILPL2. The complex is a heterotetramer with RILPL2 forming a central α-helical dimer that bridges two pRab8a molecules. The N-termini of the α-helices cross over to form an X-shaped cap (X-cap) that enables electrostatic interactions between Arg residues from RILPL2 and the phosphate moiety from pT72. RILPL2 residues in the X-cap that are critical for pRab8a binding are conserved in the RILP family of effector proteins. We find that JIP3 and JIP4 also interact specifically with LRRK2-phosphorylated Rab10, suggesting a general mode of recognition for phosphorylated Rab GTPases by phospho-specific effectors.

scholarly journals The serine/threonine kinase Par1b regulates epithelial lumen polarity via IRSp53-mediated cell–ECM signaling

2011 ◽  
Vol 192 (3) ◽  
pp. 525-540 ◽  
Author(s):  
David Cohen ◽  
Dawn Fernandez ◽  
Francisco Lázaro-Diéguez ◽  
Anne Müsch
Keyword(s):  
Mdck Cells ◽  
Adherens Junction ◽  
Cell Spreading ◽  
Effector Proteins ◽  
Fiber Formation ◽  
Scaffolding Protein ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Domain 3 ◽  
Guanosine Triphosphatase

The serine/threonine kinase Par1b promotes cell–cell adhesion and determines the polarity of the luminal domain in epithelial cells. In this study, we demonstrate that Par1b also regulates cell–extracellular matrix (ECM) signaling in kidney-derived Madin–Darby canine kidney (MDCK) cells and identified the rho–guanosine triphosphatase adaptor and scaffolding protein IRSp53 as a Par1b substrate involved in this pathway. Par1b overexpression inhibits basal lamina formation, cell spreading, focal adhesion, stress fiber formation, and compaction, whereas Par1b depletion has the opposite effect. IRSp53 depletion mimics Par1b overexpression on cell–ECM signaling and lumen polarity but had no effect on adherens junction formation. Par1b directly phosphorylates IRSp53 on S366 in cell lysates and stimulates phosphorylation on S453/3/5 via an indirect mechanism. A Par1b phosphorylation–deficient IRSp53 mutant but not the wild-type protein efficiently rescues both the cell spreading and the lumen polarity defects in Par1b MDCK cells. Our data suggest a model in which Par1b phosphorylation prevents recruitment of IRSp53 effector proteins to its Src homology domain 3 by promoting 14-3-3 binding in the vicinity of that domain.

scholarly journals Identification of a Family of Vibrio Type III Secretion System Effectors That Contain a Conserved Serine/Threonine Kinase Domain

mSphere ◽  
2021 ◽  
Author(s):  
N. Plaza ◽  
I. M. Urrutia ◽  
K. Garcia ◽  
M. K. Waldor ◽  
C. J. Blondel
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Kinase Domain ◽  
Secretion System ◽  
Effector Proteins ◽  
Serine Threonine Kinase ◽  
Content Type ◽  
Threonine Kinase ◽  
Type Iii ◽  
Infected Cells

Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function.

scholarly journals NIMA-related kinase 9–mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1

2019 ◽  
Vol 295 (5) ◽  
pp. 1240-1260 ◽  
Author(s):  
Birendra Kumar Shrestha ◽  
Mads Skytte Rasmussen ◽  
Yakubu Princely Abudu ◽  
Jack-Ansgar Bruun ◽  
Kenneth Bowitz Larsen ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Brca1 Gene ◽  
Effector Proteins ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Selective Autophagy ◽  
Sequestosome 1 ◽  
Interaction Partners ◽  
Impaired Binding

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro. A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.

scholarly journals A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18

2020 ◽  
Vol 21 (20) ◽  
pp. 7769
Author(s):  
Michal Dibus ◽  
Jan Brábek ◽  
Daniel Rösel
Keyword(s):  
Regulatory Mechanism ◽  
Negative Regulation ◽  
Effector Proteins ◽  
Terminal Part ◽  
Multiple Cancer ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Cancer Types ◽  
Feedback Regulatory Mechanism

Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.

scholarly journals Structural basis for selective targeting of Rac subfamily GTPases by a bacterial effector protein

2020 ◽  
Author(s):  
Nikolaus Balthasar Dietz ◽  
Markus Huber ◽  
Isabel Sorg ◽  
Arnaud Goepfert ◽  
Alexander Harms ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Molecular Mechanisms ◽  
Effector Proteins ◽  
Structural Basis ◽  
Effector Protein ◽  
Binding Motif ◽  
Selective Targeting ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Specific Pair

SummaryRas-homology (Rho) family GTPases are conserved molecular switches controlling fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, molecular mechanisms of target selectivity have remained elusive. Here, we report a bacterial effector protein that targets all four Rac subfamily members of Rho family GTPases, but none of the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 facilitate modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural paradigm for target selectivity towards Rac subfamily GTPases and provides a highly selective tool for their functional analysis.

scholarly journals Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

2021 ◽  
Author(s):  
Dieter Waschbüsch ◽  
Kerryn Berndsen ◽  
Axel Knebel ◽  
Dario R. Alessi ◽  
Amir R. Khan
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Specific Interaction ◽  
Structural Basis ◽  
Striking Difference ◽  
Rab Gtpases ◽  
Serine Threonine Kinase ◽  
Interaction Domain ◽  
Cellular Assays ◽  
Dependent Protein

AbstractLRRK2 serine/threonine kinase is associated with inherited Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective and closely related PPM1J/M exhibit virtually no activity toward substrates such as Rab8a phosphorylated at T72 (pT72). Here we have identified the structural determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a conserved catalytic domain that adopts a β-sandwich fold. The striking difference is that PPM1H has evolved a 110-residue flap domain that punctuates the catalytic domain. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimera with the PPM1H flap domain dephosphorylates pT72 of Rab8a with a higher specific activity than PPM1H. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.

scholarly journals Structural basis for protein glutamylation by the Legionella pseudokinase SidJ

2021 ◽  
Author(s):  
Michael Adams ◽  
Rahul Sharma ◽  
Thomas Colby ◽  
Felix Weis ◽  
Ivan Matic ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Substrate Recognition ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Effector Proteins ◽  
Structural Basis ◽  
Rab Gtpases ◽  
Binding Pockets

Legionella pneumophila (LP) avoids phagocytosis by secreting nearly 300 effector proteins into the host cytosol. SidE family of effectors (SdeA, SdeB, SdeC and SidE) employ phosphoribosyl serine ubiquitination to target an array of host Rab GTPases and innate immune factors. To suppress the deleterious toxicity of SidE enzymes in a timely manner, LP employs a metaeffector named SidJ. Upon activation by host Calmodulin (CaM), SidJ executes an ATP-dependent glutamylation to modify the catalytic residue Glu860 in the mono-ADP-ribosyl transferase (mART) domain of SdeA. SidJ is a unique glutamylase that adopts a kinase-like fold but contains two nucleotide-binding pockets. There is a lack of consensus about the substrate recognition and catalytic mechanism of SidJ. Here, we determined the cryo-EM structure of SidJ in complex with its substrate SdeA in two different states of catalysis. Our structures reveal that both phosphodiesterase (PDE) and mART domains of SdeA make extensive contacts with SidJ. In the pre-glutamylation state structure of the SidJ-SdeA complex, adenylylated E860 of SdeA is inserted into the non-canonical (migrated) nucleotide-binding pocket of SidJ. Structure-based mutational analysis indicates that SidJ employs its migrated pocket for the glutamylation of SdeA. Finally, using mass spectrometry, we identified several transient autoAMPylation sites close to both the catalytic pockets of SidJ. Our data provide unique insights into the substrate recognition and the mechanism of protein glutamylation by the pseudokinase SidJ.

scholarly journals The Serine/Threonine Kinase STK11 Promotes Shigella flexneri Dissemination through Establishment of Cell-Cell Contacts Competent for Tyrosine Kinase Signaling

2014 ◽  
Vol 82 (11) ◽  
pp. 4447-4457 ◽  
Author(s):  
Ana-Maria Dragoi ◽  
Hervé Agaisse
Keyword(s):  
Tyrosine Kinase ◽  
Shigella Flexneri ◽  
Kinase Signaling ◽  
Serine Threonine Kinase ◽  
Tyrosine Kinase Signaling ◽  
Content Dissemination ◽  
Content Type ◽  
Threonine Kinase ◽  
Membrane Protrusions ◽  
Ht 29

ABSTRACTShigella flexneriis an intracellular pathogen that disseminates in the intestinal epithelium by displaying actin-based motility. We found that althoughS. flexneridisplayed comparable actin-based motilities in the cytosols of HeLa229 and HT-29 epithelial cell lines, the overall dissemination process was much more efficient in HT-29 cells. Time-lapse microscopy demonstrated that as motile bacteria reached the cell cortex in HT-29 cells, they formed membrane protrusions that resolved into vacuoles, from which the bacteria escaped and gained access to the cytosol of adjacent cells. In HeLa229 cells,S. flexnerialso formed membrane protrusions that extended into adjacent cells, but the protrusions rarely resolved into vacuoles. Instead, the formed protrusions collapsed and retracted, bringing the bacteria back to the cytosol of the primary infected cells. Silencing the serine/threonine kinase STK11 (also known as LKB1) in HT-29 cells decreased the efficiency of protrusion resolution into vacuoles. Conversely, expressing STK11 in HeLa229 cells, which lack the STK11 locus, dramatically increased the efficiency of protrusion resolution into vacuoles.S. flexneridissemination in HT-29 cells led to the local phosphorylation of tyrosine residues in protrusions, a signaling event that was not observed in HeLa229 cells but was restored in STK11-expressing HeLa229 cells. Treatment of HT-29 cells with the tyrosine kinase inhibitor imatinib abrogated tyrosine kinase signaling in protrusions, which correlated with a severe decrease in the efficiency of protrusion resolution into vacuoles. We suggest that the formation of STK11-dependent lateral cell-cell contacts competent for tyrosine kinase signaling promotesS. flexneridissemination in epithelial cells.

scholarly journals Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1)

2021 ◽  
Vol 118 (12) ◽  
pp. e2023245118
Author(s):  
Nikolaus Dietz ◽  
Markus Huber ◽  
Isabel Sorg ◽  
Arnaud Goepfert ◽  
Alexander Harms ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Molecular Switches ◽  
Small Gtpases ◽  
Bacterial Toxins ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Structural Basis ◽  
Effector Protein ◽  
Rho Family ◽  
Specific Pair

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

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