scholarly journals An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

2017 ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Daniel W. Buster ◽  
Brian J. Galletta ◽  
Cody J. Boese ◽  
John M. Ryniawec ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Early Step ◽  
Centriole Duplication ◽  
Physiological Relevance ◽  
Phosphorylation Pattern ◽  
Centriole Assembly ◽  
Assembly Site

AbstractPolo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event, involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 binding stimulates Plk4 kinase activity in vitro, and, in turn, is phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STAN domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N–terminus of Ana2 which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

scholarly journals An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

2018 ◽  
Vol 217 (4) ◽  
pp. 1217-1231 ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Daniel W. Buster ◽  
Brian J. Galletta ◽  
Cody J. Boese ◽  
John M. Ryniawec ◽  
...  
Keyword(s):  
Central Region ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Early Step ◽  
Centriole Duplication ◽  
Physiological Relevance ◽  
N Terminus ◽  
Phosphorylation Pattern ◽  
Centriole Assembly ◽  
Assembly Site

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4–Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

scholarly journals The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Matthew A Cottee ◽  
Nadine Muschalik ◽  
Steven Johnson ◽  
Joanna Leveson ◽  
Jordan W Raff ◽  
...  
Keyword(s):  
Point Mutations ◽  
Coiled Coil ◽  
Higher Order ◽  
Centriole Duplication ◽  
Terminal Domain ◽  
Coiled Coil Domain ◽  
Centriole Assembly ◽  
Tetramer Structure

Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity

2009 ◽  
Vol 419 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Amber L. Couzens ◽  
Vivian Saridakis ◽  
Michael P. Scheid
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Agc Kinases ◽  
Threonine Residue ◽  
Substrate Phosphorylation ◽  
Hydrophobic Motif

ROCK (Rho-associated coiled-coil kinase) 2 is a member of the AGC kinase family that plays an essential role downstream of Rho in actin cytoskeleton assembly and contractility. The process of ROCK2 activation is complex and requires suppression of an autoinhibitory mechanism that is facilitated by Rho binding. ROCK2 harbours a C-terminal extension within the kinase domain that contains a hydrophobic cluster of phenylalanine and tyrosine residues surrounding a key threonine residue. In growth-factor-stimulated AGC kinases, the hydrophobic motif is important for the transition of the kinase from inactive to active complex and requires phosphorylation of the conserved serine/threonine residue. Less is understood about the contribution that the hydrophobic motif plays in the activation of ROCK, and the role of the hydrophobic motif threonine at position 405. In the present study, we show that this residue of ROCK is essential for substrate phosphorylation and kinase domain dimerization. However, in contrast with the growth-factor-activated AGC kinases, a phosphomimetic residue at position 405 was inhibitory for ROCK2 activity and dimerization. A soluble hydrophobic motif peptide allosterically activated ROCK2 In vitro, but not the equivalent peptide with Asp405 substitution. Mechanistically, both ROCK2 activity and dimerization were dependent upon the interaction between Thr405 of the hydrophobic motif and Asp39 of the N-terminal extension. The reciprocal exchange of these residues was permissive for kinase activity, but dimerization was lost. These results support the rationale for development of small-molecule inhibitors designed to block ROCK activation by selectively interfering with hydrophobic motif-mediated activation-state transition and dimer formation.

scholarly journals Activity of the Ste20-like kinase, SLK, is enhanced by homodimerization

2011 ◽  
Vol 301 (3) ◽  
pp. F554-F564 ◽  
Author(s):  
Sierra Delarosa ◽  
Julie Guillemette ◽  
Joan Papillon ◽  
Ying-Shan Han ◽  
Arnold S. Kristof ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Gel Filtration ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Luciferase Reporter ◽  
Fk506 Binding Protein ◽  
Induced Apoptosis ◽  
Injury And Repair

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1–373) and a modified FK506 binding protein, Fv (Fv-SLK 1–373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1–373. In an in vitro kinase assay, the dimeric Fv-SLK 1–373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1–373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1–373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1–373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.

Nuclear receptor-binding protein 1: a novel tumour suppressor and pseudokinase

2013 ◽  
Vol 41 (4) ◽  
pp. 1055-1060 ◽  
Author(s):  
Jason S. Kerr ◽  
Catherine H. Wilson
Keyword(s):  
Nuclear Receptor ◽  
Receptor Binding ◽  
Kinase Activity ◽  
Binding Protein ◽  
Kinase Domain ◽  
Present Review ◽  
Critical Components ◽  
Receptor Binding Protein

Pseudokinases are a class of kinases which are structurally designated as lacking kinase activity. Despite the lack of kinase domain sequence conservation, there is increasing evidence that a number of pseudokinases retain kinase activity and/or have critical cellular functions, casting aside previous notions that pseudokinases simply exist as redundant kinases. Moreover, a number of recent studies have implicated pseudokinases as critical components in cancer formation and progression. The present review discusses the interactions and potential functions that nuclear receptor-binding protein 1, a pseudokinase recently described to have a tumour-suppressive role in cancer, may play in cellular homoeostasis and protein regulation. The recent findings highlighted in the present review emphasize the requirement to fully determine the function of pseudokinases in vitro and in vivo, the understanding of which may ultimately uncover new directions for drug discovery.

scholarly journals Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97

2007 ◽  
Vol 88 (2) ◽  
pp. 395-404 ◽  
Author(s):  
Vera Schregel ◽  
Sabrina Auerochs ◽  
Ramona Jochmann ◽  
Katja Maurer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Interaction Domain ◽  
Amino Acid Region ◽  
Substrate Phosphorylation ◽  
Regulatory Functions ◽  
Acid Region

The human cytomegalovirus-encoded protein kinase pUL97 is a determinant of efficient virus replication and fulfils several regulatory functions. In particular, pUL97 interacts with and phosphorylates viral and cellular proteins. Substrate phosphorylation has regulatory consequences on viral replicative stages such as DNA synthesis, transcription and nuclear capsid egress. pUL97, in accordance with related herpesviral protein kinases, possesses strong autophosphorylation activity. Here, we demonstrate that pUL97 shows a pronounced potential to self-interact. Self-interaction of pUL97 is not dependent on its kinase activity, as seen with a catalytically inactive point mutant. The property of self-interaction maps to the amino acid region 231–280 which is separable from the postulated kinase domain. The detection of high-molecular-mass complexes of pUL97 suggests the formation of dimers and oligomers. Importantly, the analysis of pUL97 mutants by in vitro kinase assays demonstrated a correlation between self-interaction and protein kinase activity, i.e. all mutants lacking the ability to self-interact were negative or reduced in their kinase activity. Thus, our findings provide novel insights into the pUL97 structure–activity relationship suggesting an importance of self-interaction for pUL97 functionality.

Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 438-438 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Kyi Min Saw ◽  
Allen Eaves ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

scholarly journals Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110012 ◽  
Author(s):  
Helen I. Woodroof ◽  
Joe H. Pogson ◽  
Mike Begley ◽  
Lewis C. Cantley ◽  
Maria Deak ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Substrate Specificity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Missense Mutations ◽  
Pediculus Humanus ◽  
Tensin Homolog

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro . We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated—namely Drosophila melanogaster (dPINK1) , Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)—are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.

scholarly journals Negative regulation of Raf-1 by phosphorylation of serine 621.

1996 ◽  
Vol 16 (10) ◽  
pp. 5409-5418 ◽  
Author(s):  
H Mischak ◽  
T Seitz ◽  
P Janosch ◽  
M Eulitz ◽  
H Steen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Negative Regulation ◽  
Catalytic Function ◽  
Dependent Protein ◽  
The One

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

scholarly journals Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4

2017 ◽  
Vol 114 (5) ◽  
pp. E879-E886 ◽  
Author(s):  
Maria Castañeda-Bueno ◽  
Juan Pablo Arroyo ◽  
Junhui Zhang ◽  
Jeremy Puthumana ◽  
Orlando Yarborough ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cultured Cells ◽  
Kinase Domain ◽  
Tandem Mass ◽  
Volume Depletion ◽  
Kinase Activation ◽  
Downstream Signaling ◽  
Electrolyte Homeostasis

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

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