scholarly journals N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization

2008 ◽  
Vol 411 (2) ◽  
pp. 407-414 ◽  
Author(s):  
Ritu Garg ◽  
Kirsi Riento ◽  
Nicholas Keep ◽  
Jonathan D. H. Morris ◽  
Anne J. Ridley
Keyword(s):  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Serine Threonine Kinase ◽  
Actin Reorganization ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Coiled Coil Region ◽  
N Terminus ◽  
In Cells

ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I1–420 is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE.

scholarly journals Intermolecular and Intramolecular Interactions Regulate Catalytic Activity of Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase α

2001 ◽  
Vol 21 (8) ◽  
pp. 2767-2778 ◽  
Author(s):  
Ivan Tan ◽  
Kah Tong Seow ◽  
Louis Lim ◽  
Thomas Leung
Keyword(s):  
Myotonic Dystrophy ◽  
Phorbol Ester ◽  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Intramolecular Interactions ◽  
Serine Threonine Kinase ◽  
Kinase Activation ◽  
Rich Domain ◽  
N Terminus

ABSTRACT Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKα to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKα multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKα catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase–distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation.

scholarly journals Domains of WNK1 kinase in the regulation of ROMK1

2008 ◽  
Vol 295 (2) ◽  
pp. F438-F445 ◽  
Author(s):  
Hao-Ran Wang ◽  
Zhen Liu ◽  
Chou-Long Huang
Keyword(s):  
Amino Acids ◽  
Potassium Channel ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Autoinhibitory Domain ◽  
Rich Domain ◽  
Coiled Coil Domain ◽  
Necessary And Sufficient

WNK1 kinase belongs to a family of serine-threonine protein kinases with an atypical placement of the catalytic lysine. Increased expression of WNK1 causes hypertension and hyperkalemia in humans. WNK1 inhibits renal potassium channel ROMK1 by enhancing its endocytosis, likely contributing to hyperkalemia in affected patients. The domains of WNK1 involved in inhibition of ROMK1 have not been completely elucidated. Here, we reported that an NH2-terminal proline-rich domain (N-PRD; amino acids 1-119) is necessary and sufficient for WNK1 inhibition of ROMK1. A region (named “NL” for N-linker; amino acids 120-220) located between N-PRD and the kinase domain of WNK1 (amino acids 220-491) antagonized the inhibition of ROMK1 caused by N-PRD. The WNK1 kinase domain reversed the antagonism of NL on N-PRD. Mutagenesis studies revealed that charge-charge interactions between two conserved catalytic residues (Lys-233 and Asp-368) within the kinase domain (not the kinase activity) are critical for kinase domain to reverse the antagonism of NL domain. The WNK1 autoinhibitory domain (AID; amino acids 491-555) also affected ROMK, presumably by modulating the kinase domain conformation. Mutations of two conserved phenylalanine abolished the ability of AID to modulate ROMK1. Finally, the first coiled-coil domain (CC1; amino acids 555-640) of WNK1 alleviated the effect of AID domain toward kinase domain. Thus, multiple intra- and/or intermolecular interactions of WNK1 domains are at play for regulation of ROMK1 by WNK1.

scholarly journals Phosphorylation of Serine 114 on Atg32 mediates mitophagy

2011 ◽  
Vol 22 (17) ◽  
pp. 3206-3217 ◽  
Author(s):  
Yoshimasa Aoki ◽  
Tomotake Kanki ◽  
Yuko Hirota ◽  
Yusuke Kurihara ◽  
Tetsu Saigusa ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Quality Control ◽  
Significant Role ◽  
Coiled Coil ◽  
Adaptor Protein ◽  
Mitochondrial Quality Control ◽  
Signal Transduction Cascade ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
N Terminus

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When mitophagy is induced in yeast, mitochondrial residential protein Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and it is recruited into the vacuole along with mitochondria. The Atg11–Atg32 interaction is believed to be the initial molecular step in which the autophagic machinery recognizes mitochondria as a cargo, although how this interaction is mediated is poorly understood. Therefore, we studied the Atg11–Atg32 interaction in detail. We found that the C-terminus region of Atg11, which included the fourth coiled-coil domain, interacted with the N-terminus region of Atg32 (residues 100–120). When mitophagy was induced, Ser-114 and Ser-119 on Atg32 were phosphorylated, and then the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediated the Atg11–Atg32 interaction and mitophagy. These findings suggest that cells can regulate the amount of mitochondria, or select specific mitochondria (damaged or aged) that are degraded by mitophagy, by controlling the activity and/or localization of the kinase that phosphorylates Atg32. We also found that Hog1 and Pbs2, which are involved in the osmoregulatory signal transduction cascade, are related to Atg32 phosphorylation and mitophagy.

scholarly journals Expression, purification and crystallization of the SKICH domain of human TAX1BP1

2014 ◽  
Vol 70 (5) ◽  
pp. 619-623 ◽  
Author(s):  
Yang Yang ◽  
Guan Wang ◽  
Xiaolan Huang ◽  
Zhihua Du
Keyword(s):  
Coiled Coil ◽  
Molecular Replacement ◽  
Data Set ◽  
C Terminus ◽  
Zinc Finger Motifs ◽  
Coiled Coil Region ◽  
Ubiquitin Binding ◽  
N Terminus ◽  
Replacement Solution

TAX1BP1 is a highly conserved, pleiotropic protein that plays many essential functions in human cells, including negative regulation of inflammatory and antimicrobial responses mediated by NF-κB and IRF3 signaling, inhibition of apoptosis, transcriptional coactivation and autophagyetc.TAX1BP1 contains a SKICH domain at the N-terminus, three coiled-coil domains in the middle and two ubiquitin-binding zinc-finger motifs at the C-terminus. The SKICH domain and the linker sequence between the SKICH domain and the coiled-coil region mediate interaction with ubiquitin-like proteins of the LC3/GABARAP family, which are autophagosome markers. For structure determination of the SKICH domain of TAX1BP1, a protein construct (amino acids 15–148) corresponding to the SKICH domain plus the linker region was expressed, purified and crystallized. A native diffraction data set has been collected to 1.9 Å resolution. A molecular-replacement solution has been found by using the structure of the SKICH domain of NDP52, a paralog of TAX1BP1.

scholarly journals Cep57, a multidomain protein with unique microtubule and centrosomal localization domains

2008 ◽  
Vol 412 (2) ◽  
pp. 265-273 ◽  
Author(s):  
Ko Momotani ◽  
Alexander S. Khromov ◽  
Tsuyoshi Miyake ◽  
P. Todd Stukenberg ◽  
Avril V. Somlyo
Keyword(s):  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Centrosomal Protein ◽  
Multidomain Protein ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
N Terminus

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to γ-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.

scholarly journals Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state

2018 ◽  
Vol 29 (23) ◽  
pp. 2874-2886 ◽  
Author(s):  
Cody J. Boese ◽  
Jonathan Nye ◽  
Daniel W. Buster ◽  
Tiffany A. McLamarrah ◽  
Amy E. Byrnes ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Dominant Role ◽  
Kinase Domain ◽  
Feedback Mechanism ◽  
Phosphorylation State ◽  
Negative Feedback Mechanism ◽  
Centriole Duplication ◽  
C Terminus ◽  
N Terminus ◽  
Opposing Effects

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole “targeting-factor.” In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4’s kinase domain. Thus, Asl-A’s phosphorylation state determines which of Asl-A’s two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

scholarly journals ClpB in a Cyanobacterium: Predicted Structure, Phylogenetic Relationships, and Regulation by Light and Temperature

1998 ◽  
Vol 180 (19) ◽  
pp. 5173-5182 ◽  
Author(s):  
Martina Celerin ◽  
Andrea A. Gilpin ◽  
Nicholas J. Schisler ◽  
Alexander G. Ivanov ◽  
Ewa Miskiewicz ◽  
...  
Keyword(s):  
Phylogenetic Relationships ◽  
Stress Protein ◽  
Coiled Coil ◽  
Genomic Clone ◽  
Optimal Conditions ◽  
Coiled Coil Domain ◽  
Coiled Coil Region ◽  
Putative Site ◽  
Phosphorylation Domain ◽  
In Cells

ABSTRACT The sequence of a genomic clone encoding a 100-kDa stress protein of Plectonema boryanum (p-ClpB) was determined. The predicted polypeptide contains two putative ATPase regions located within two highly conserved domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conserved consensus CK2 phosphorylation domain. The coiled-coil region and the putative site of phosphorylation are not unique to p-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogs ClpA, ClpC, ClpX, and ClpY. Small quantities of a 4.5-kbp-clpB transcript and 110-kDa cytosolic p-ClpB protein were detected in cells grown under optimal conditions; however, increases in the quantities of the transcript and protein were observed in cells grown under excess light and low temperature conditions. Finally, we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationships among the homologs. We have used this information, along with an identity alignment, to redefine the Clp subfamilies.

The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2055-2068 ◽  
Author(s):  
Daniel V. Zurawski ◽  
Murry A. Stein
Keyword(s):  
Type Iii Secretion ◽  
Coiled Coil ◽  
Amphipathic Helix ◽  
Yeast Two Hybrid ◽  
C Terminus ◽  
N Terminus ◽  
Domain Mapping ◽  
Self Association ◽  
Discrete Domains ◽  
Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

scholarly journals Tyr198 is the Essential Autophosphorylation Site for STK16 Localization and Kinase Activity

2019 ◽  
Vol 20 (19) ◽  
pp. 4852 ◽  
Author(s):  
Junjun Wang ◽  
Juanjuan Liu ◽  
Xinmiao Ji ◽  
Xin Zhang
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Serine Threonine Kinase ◽  
Cycle Progression ◽  
Threonine Kinase ◽  
Cellular Processes ◽  
Activation Segment ◽  
And Function

STK16, reported as a Golgi localized serine/threonine kinase, has been shown to participate in multiple cellular processes, including the TGF-β signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. However, the mechanisms of the regulation of its kinase activity remain underexplored. It was known that STK16 is autophosphorylated at Thr185, Ser197, and Tyr198 of the activation segment in its kinase domain. We found that STK16 localizes to the cell membrane and the Golgi throughout the cell cycle, but mutations in the auto-phosphorylation sites not only alter its subcellular localization but also affect its kinase activity. In particular, the Tyr198 mutation alone significantly reduced the kinase activity of STK16, abolished its Golgi and membrane localization, and affected the cell cycle progression. This study demonstrates that a single site autophosphorylation of STK16 could affect its localization and function, which provides insights into the molecular regulatory mechanism of STK16’s kinase activity.

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