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

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.

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Par1b links lumen polarity with LGN–NuMA positioning for distinct epithelial cell division phenotypes

The Journal of Cell Biology ◽

10.1083/jcb.201303013 ◽

2013 ◽

Vol 203 (2) ◽

pp. 251-264 ◽

Cited By ~ 27

Author(s):

Francisco Lázaro-Diéguez ◽

David Cohen ◽

Dawn Fernandez ◽

Louis Hodgson ◽

Sven C.D. van IJzendoorn ◽

...

Keyword(s):

Hepg2 Cells ◽

Mdck Cells ◽

Rho Kinase ◽

Cell Contact ◽

Cleavage Furrow ◽

Mitotic Spindles ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Rhoa Activity ◽

Columnar Organization

Columnar epithelia establish their luminal domains and their mitotic spindles parallel to the basal surface and undergo symmetric cell divisions in which the cleavage furrow bisects the apical domain. Hepatocyte lumina interrupt the lateral domain of neighboring cells perpendicular to two basal domains and their cleavage furrow rarely bifurcates the luminal domains. We determine that the serine/threonine kinase Par1b defines lumen position in concert with the position of the astral microtubule anchoring complex LGN–NuMA to yield the distinct epithelial division phenotypes. Par1b signaling via the extracellular matrix (ECM) in polarizing cells determined RhoA/Rho-kinase activity at cell–cell contact sites. Columnar MDCK and Par1b-depleted hepatocytic HepG2 cells featured high RhoA activity that correlated with robust LGN–NuMA recruitment to the metaphase cortex, spindle alignment with the substratum, and columnar organization. Reduced RhoA activity at the metaphase cortex in HepG2 cells and Par1b-overexpressing MDCK cells correlated with a single or no LGN–NuMA crescent, tilted spindles, and the development of lateral lumen polarity.

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Apical surface formation in MDCK cells: regulation by the serine/threonine kinase EMK1

Methods ◽

10.1016/s1046-2023(03)00033-1 ◽

2003 ◽

Vol 30 (3) ◽

pp. 269-276 ◽

Cited By ~ 23

Author(s):

David Cohen ◽

Anne Müsch

Keyword(s):

Mdck Cells ◽

Apical Surface ◽

Serine Threonine Kinase ◽

Surface Formation ◽

Threonine Kinase

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

mSphere ◽

10.1128/msphere.00599-21 ◽

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.

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Gα12 regulates epithelial cell junctions through Src tyrosine kinases

AJP Cell Physiology ◽

10.1152/ajpcell.00548.2002 ◽

2003 ◽

Vol 285 (5) ◽

pp. C1281-C1293 ◽

Cited By ~ 42

Author(s):

Tobias N. Meyer ◽

Jennifer Hunt ◽

Catherine Schwesinger ◽

Bradley M. Denker

Keyword(s):

Epithelial Cell ◽

Tyrosine Kinase ◽

Mdck Cells ◽

Adherens Junction ◽

Paracellular Permeability ◽

Cell Junctions ◽

Scaffolding Protein ◽

Junctional Complex ◽

Adherens Junction Proteins ◽

Junction Proteins

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Gα12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Gα12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Gα12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Gα12 (QLα12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLα12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLα12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLα12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLα12-expressing MDCK cells as assessed by Src autophosphorylation, and β-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Gα12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010068 ◽

2019 ◽

Vol 295 (5) ◽

pp. 1240-1260 ◽

Cited By ~ 3

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.

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A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18

International Journal of Molecular Sciences ◽

10.3390/ijms21207769 ◽

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.

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The mTORC1/S6K/PDCD4/eIF4A axis determines outcome of mitosis

10.1101/794545 ◽

2019 ◽

Author(s):

Mohamed Moustafa-Kamal ◽

Thomas Kucharski ◽

Wissal El Assad ◽

Valentina Gandin ◽

Yazan Abas ◽

...

Keyword(s):

Cell Death ◽

Normal Cell ◽

Scaffolding Protein ◽

Tumor Suppressor Protein ◽

Dramatic Reduction ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Cell Growth And Proliferation ◽

Mtor Complex 1 ◽

Cell Death Protein

AbstractmTOR is a serine/threonine kinase which acts a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in mitotic arrested cells across a variety of cancer and normal cell lines. Prevention of raptor phosphorylation during mitosis resulted in reactivation of mTORC1 in a rapamycin-sensitive manner. Importantly, expression of a non-phosphorylatable raptor mutant caused a dramatic reduction in cytotoxicity of the spindle poison Taxol. This effect was mediated via degradation of Programmed Cell Death Protein 4 (PDCD4), a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A was able to enhance the effects of taxol and restore sensitivity in Taxol resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in death vs. slippage decision during prolonged mitotic arrest and may be exploited to gain a clinical benefit in treating cancers resistant to anti-mitotic drugs.

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

10.1101/739813 ◽

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.

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