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

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.

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

Biochemical Journal ◽

10.1042/bj20071342 ◽

2008 ◽

Vol 411 (2) ◽

pp. 407-414 ◽

Cited By ~ 17

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.

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A molecular mechanism for the procentriole recruitment of Ana2

10.1101/647149 ◽

2019 ◽

Author(s):

Tiffany A. McLamarrah ◽

Sarah K. Speed ◽

Daniel W. Buster ◽

Carey J. Fagerstrom ◽

Brian J. Galletta ◽

...

Keyword(s):

Molecular Mechanism ◽

Kinase Activity ◽

Surface Protein ◽

Single Site ◽

Centriole Duplication ◽

C Terminus ◽

N Terminus ◽

Mother Centriole ◽

Centriole Assembly ◽

Daughter Centriole

AbstractCentriole duplication begins with the assembly of a pre-procentriole at a single site on a mother centriole and proceeds with the hierarchical recruitment of a conserved set of proteins, including Polo-like kinase 4 (Plk4)/ZYG-1, Ana2/SAS-5/STIL, and the cartwheel protein Sas6. During assembly, Ana2/STIL stimulates Plk4 kinase activity, and in turn, Ana2/STIL’s C-terminus is phosphorylated, allowing it to bind and recruit Sas6. The assembly steps immediately preceding Sas6-loading appear clear, but the mechanism underlying the upstream pre-procentriole recruitment of Ana2/STIL is not. In contrast to proposed models of Ana2/STIL recruitment, we recently showed that Drosophila Ana2 targets procentrioles independent of Plk4-binding. Instead, Ana2 recruitment requires Plk4 phosphorylation of Ana2’s N-terminus, but the mechanism explaining this process is unknown. Here, we show that the amyloid-like domain of Sas4, a centriole surface protein, binds Plk4 and Ana2, and facilitates phosphorylation of Ana2’s N-terminus which increases Ana2’s affinity for Sas4. Consequently, Ana2 accumulates at the procentriole to induce daughter centriole assembly.

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Regulation of Jak2 Function by Phosphorylation of Tyr317 and Tyr637 during Cytokine Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00278-09 ◽

2009 ◽

Vol 29 (12) ◽

pp. 3367-3378 ◽

Cited By ~ 23

Author(s):

Scott A. Robertson ◽

Rositsa I. Koleva ◽

Lawrence S. Argetsinger ◽

Christin Carter-Su ◽

Jarrod A. Marto ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Feedback Inhibition ◽

Dominant Role ◽

Kinase Domain ◽

Cytokine Signaling ◽

Tyrosine Kinase Domain ◽

Phosphorylation Sites ◽

Cytokine Stimulation

ABSTRACT Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.

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Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1

Eukaryotic Cell ◽

10.1128/ec.00291-10 ◽

2011 ◽

Vol 10 (3) ◽

pp. 313-319 ◽

Cited By ~ 19

Author(s):

Yang Liu ◽

Xinjing Xu ◽

Marian Carlson

Keyword(s):

Protein Kinase ◽

Kinase Domain ◽

Catalytic Subunit ◽

Glucose Limitation ◽

Content Type ◽

Snf1 Kinase ◽

Phosphorylation State ◽

Glucose Depletion ◽

C Terminus ◽

Amp Activated Protein Kinase

ABSTRACT The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.

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N-terminal mutations modulate yeast SNF1 protein kinase function.

Genetics ◽

10.1093/genetics/132.3.639 ◽

1992 ◽

Vol 132 (3) ◽

pp. 639-650 ◽

Cited By ~ 12

Author(s):

F Estruch ◽

M A Treitel ◽

X Yang ◽

M Carlson

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Phosphorylation Site ◽

Gel Filtration ◽

Glucose Deprivation ◽

Kinase Domain ◽

Snf1 Kinase ◽

N Protein ◽

N Terminus

Abstract The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-delta N kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-delta N protein showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell.

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A molecular mechanism for the procentriole recruitment of Ana2

The Journal of Cell Biology ◽

10.1083/jcb.201905172 ◽

2019 ◽

Vol 219 (2) ◽

Cited By ~ 1

Author(s):

Tiffany A. McLamarrah ◽

Sarah K. Speed ◽

John M. Ryniawec ◽

Daniel W. Buster ◽

Carey J. Fagerstrom ◽

...

Keyword(s):

Molecular Mechanism ◽

Kinase Activity ◽

Outer Wall ◽

Single Site ◽

Structural Motif ◽

Centriole Duplication ◽

Short Region ◽

N Terminus ◽

Mother Centriole ◽

Daughter Centriole

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2–G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.

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Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

The Journal of Cell Biology ◽

10.1083/jcb.201410105 ◽

2015 ◽

Vol 208 (4) ◽

pp. 401-414 ◽

Cited By ~ 26

Author(s):

Joseph E. Klebba ◽

Brian J. Galletta ◽

Jonathan Nye ◽

Karen M. Plevock ◽

Daniel W. Buster ◽

...

Keyword(s):

Cell Cycle ◽

Drosophila Melanogaster ◽

Binding Domain ◽

Terminal Region ◽

Scaffolding Protein ◽

Binding Partner ◽

Centriole Duplication ◽

Binding Domains ◽

C Terminus ◽

N Terminus

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.

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Distinct properties of Ca2+–calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel

The Journal of General Physiology ◽

10.1085/jgp.201210810 ◽

2012 ◽

Vol 140 (5) ◽

pp. 541-555 ◽

Cited By ~ 63

Author(s):

Sze-Yi Lau ◽

Erik Procko ◽

Rachelle Gaudet

Keyword(s):

Binding Site ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Feedback Mechanism ◽

Sequence Motif ◽

Recognition Sequence ◽

Calmodulin Binding ◽

Negative Feedback Mechanism ◽

C Terminus ◽

Wide Range

Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca2+–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca2+–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca2+–CaM, respectively. This structure is similar to canonical Ca2+–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca2+–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca2+–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca2+–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.

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Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain

Biochemical Journal ◽

10.1042/bj3570513 ◽

2001 ◽

Vol 357 (2) ◽

pp. 513-520 ◽

Cited By ~ 2

Author(s):

Zheng ZHU ◽

Song LING ◽

Qi-Heng YANG ◽

Lin LI

Keyword(s):

Kinase Activity ◽

Ph Dependence ◽

Critical Role ◽

Inhibitory Effect ◽

Kinase Domain ◽

Wild Type ◽

Wild Type Enzyme ◽

Dependent Protein Kinase ◽

C Terminus ◽

Dependent Protein

The bisphosphatase activity of the hepatic bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is repressed by its kinase domain, and regulated by cAMP-dependent protein kinase (PKA)-catalysed phosphorylation. In the present study, the mechanism by which the bisphosphatase activity is repressed by the kinase domain and regulated by phosphorylation was investigated. We found that truncation of the C-terminus of the enzyme by 25, but not 20, amino acids dramatically enhanced the catalytic rate of the bisphosphatase, abrogated the inhibition by the kinase domain, and eliminated the effect of PKA-mediated phosphorylation on activity. In addition, mutation of His444-Arg-Glu-Arg to Ala-Ala-Glu-Ala had similar effects as the deletion. Moreover, the mutations also significantly affected the phosphorylation-mediated regulation of the kinase activity of the enzyme. Furthermore, the mutations altered the pH-dependence of the bisphosphatase, and the mutant bisphosphatases were more sensitive to modification by diethyl pyrocarbonate and guanidine-induced inactivation than the wild-type enzyme. Taken together, these results demonstrate that the sequence His444-Arg-Glu-Arg plays a critical role in repression of the bisphosphatase activity by both the N-terminal kinase domain and the C-terminal tail itself. These results also explain the activation of the bisphosphatase activity by PKA-catalysed phosphorylation, by suggesting that phosphorylation may relieve the inhibitory effect of the kinase domain that is mediated by the three basic residues in this sequence.

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

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2767-2778.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2767-2778 ◽

Cited By ~ 53

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.

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