scholarly journals TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development

2011 ◽  
Vol 437 (1) ◽  
pp. 157-167 ◽  
Author(s):  
Michale Bouskila ◽  
Noor Esoof ◽  
Laurie Gay ◽  
Emily H. Fang ◽  
Maria Deak ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protein Expression ◽  
Spinocerebellar Ataxia ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Spinocerebellar Ataxia Type ◽  
Protein Kinase Activity ◽  
Kinase Substrate ◽  
The Impact

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

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 LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity

2007 ◽  
Vol 405 (2) ◽  
pp. 307-317 ◽  
Author(s):  
Mahaboobi Jaleel ◽  
R. Jeremy Nichols ◽  
Maria Deak ◽  
David G. Campbell ◽  
Frank Gillardon ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Late Onset ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Kinase Substrate ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.

scholarly journals Pie1, a Protein Interacting with Mec1, Controls Cell Growth and Checkpoint Responses in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (3) ◽  
pp. 755-764 ◽  
Author(s):  
Tatsushi Wakayama ◽  
Tae Kondo ◽  
Seiko Ando ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Damage ◽  
Cell Growth ◽  
Kinase Activity ◽  
Critical Role ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Replication Checkpoint ◽  
Family Protein ◽  
Replication Block

ABSTRACT In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Δ mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Δ cells, as in mec1Δcells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of thepie1Δ mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Δ mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

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.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

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 Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining

1999 ◽  
Vol 19 (5) ◽  
pp. 3877-3884 ◽  
Author(s):  
Akihiro Kurimasa ◽  
Satoshi Kumano ◽  
Nikolai V. Boubnov ◽  
Michael D. Story ◽  
Chang-Shung Tung ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Human Dna ◽  
Dna Dsbs ◽  
Dependent Protein ◽  
Dna Strand

ABSTRACT The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

scholarly journals p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2Cδ

2004 ◽  
Vol 382 (2) ◽  
pp. 425-431 ◽  
Author(s):  
Ulrik DOEHN ◽  
Steen GAMMELTOFT ◽  
Shi-Hsiang SHEN ◽  
Claus J. JENSEN
Keyword(s):  
Protein Phosphatase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Dependent Manner ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase 2 ◽  
P90 Ribosomal S6 Kinase ◽  
Ribosomal S6 Kinase

RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cδ (protein phosphatase 2Cδ). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cδ is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cδ(H154D) (His154→Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1–71 of PP2Cδ and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cδ associates with RSK1–RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cδ in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.

scholarly journals Mutational Activation of ErbB2 Reveals a New Protein Kinase Autoinhibition Mechanism

2007 ◽  
Vol 283 (3) ◽  
pp. 1588-1596 ◽  
Author(s):  
Ying-Xin Fan ◽  
Lily Wong ◽  
Jinhui Ding ◽  
Nikolay A. Spiridonov ◽  
Richard C. Johnson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Human Cancer ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Mitogen Activated Protein ◽  
Cancer Mutations ◽  
Mutational Activation ◽  
New Protein

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the αC helix and β4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to ∼10- and ∼7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the αC-β4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the αC-β4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the αC-β4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of αC-β4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

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