Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function

Centrins are a family of small, calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified centrin 2 and centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, although Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole overproduction, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, whereas depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr-676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes.

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AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005040 ◽

2018 ◽

Vol 293 (46) ◽

pp. 17853-17862 ◽

Cited By ~ 13

Author(s):

Samaneh Mafakheri ◽

Ralf R. Flörke ◽

Sibylle Kanngießer ◽

Sonja Hartwig ◽

Lena Espelage ◽

...

Keyword(s):

Catalytic Activity ◽

Protein Kinase ◽

Full Length ◽

Rab Gtpase ◽

Phosphorylation Sites ◽

Rab Gtpases ◽

Genetic Ablation ◽

Regulatory Domains ◽

Amp Activated Protein Kinase

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

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Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1

Molecular Biology of the Cell ◽

10.1091/mbc.e15-07-0527 ◽

2016 ◽

Vol 27 (6) ◽

pp. 1040-1050 ◽

Cited By ~ 19

Author(s):

Shenshen Lai ◽

Steven Pelech

Keyword(s):

Catalytic Activity ◽

Protein Kinase ◽

Protein Kinases ◽

Variable Region ◽

Mitogen Activated Protein Kinase ◽

General Mechanism ◽

Common Mechanism ◽

Phosphorylation Sites ◽

Eukaryotic Protein

The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Their phosphorylation within the well-known activation T-loop, a variable region between protein kinase catalytic subdomains VII and VIII, is a common mechanism for stimulation of their phosphotransferase activities. Extracellular signal–regulated kinase 1 (ERK1), a member of the extensively studied mitogen-activated protein kinase (MAPK) family, serves as a paradigm for regulation of protein kinases in signaling modules. In addition to the well-documented T202 and Y204 stimulatory phosphorylation sites in the activation T-loop of ERK1 and its closest relative, ERK2, three additional flanking phosphosites have been confirmed (T198, T207, and Y210 from ERK1) by high-throughput mass spectrometry. In vitro kinase assays revealed the functional importance of T207 and Y210, but not T198, in negatively regulating ERK1 catalytic activity. The Y210 site could be important for proper conformational arrangement of the active site, and a Y210F mutant could not be recognized by MEK1 for phosphorylation of T202 and Y204 in vitro. Autophosphorylation of T207 reduces the catalytic activity and stability of activated ERK1. We propose that after the activation of ERK1 by MEK1, subsequent slower phosphorylation of the flanking sites results in inhibition of the kinase. Because the T207 and Y210 phosphosites of ERK1 are highly conserved within the eukaryotic protein kinase family, hyperphosphorylation within the kinase activation T-loop may serve as a general mechanism for protein kinase down-regulation after initial activation by their upstream kinases.

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Gene structure and expression of an unusual protein kinase from Plasmodium falciparum homologous at its carboxyl terminus with the EF hand calcium-binding proteins.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53616-4 ◽

1993 ◽

Vol 268 (6) ◽

pp. 4347-4354

Author(s):

Y. Zhao ◽

B. Kappes ◽

R.M. Franklin

Keyword(s):

Plasmodium Falciparum ◽

Protein Kinase ◽

Gene Structure ◽

Calcium Binding ◽

Binding Proteins ◽

Calcium Binding Proteins ◽

Carboxyl Terminus ◽

Ef Hand ◽

Gene Structure And Expression

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cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36652-8 ◽

1994 ◽

Vol 269 (20) ◽

pp. 14509-14517 ◽

Cited By ~ 8

Author(s):

E. Butt ◽

K. Abel ◽

M. Krieger ◽

D. Palm ◽

V. Hoppe ◽

...

Keyword(s):

Protein Kinase ◽

Focal Adhesion ◽

Human Platelets ◽

Phosphorylation Sites ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Kinase Phosphorylation ◽

Camp And Cgmp

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Neural network dysfunction in bipolar depression: clues from the efficacy of lamotrigine

Biochemical Society Transactions ◽

10.1042/bst0371080 ◽

2009 ◽

Vol 37 (5) ◽

pp. 1080-1084 ◽

Cited By ~ 12

Author(s):

Charles H. Large ◽

Elena Di Daniel ◽

Xingbao Li ◽

Mark S. George

Keyword(s):

Bipolar Disorder ◽

Valproic Acid ◽

Protein Kinase ◽

Glycogen Synthase ◽

Bipolar Depression ◽

Inhibitory Effect ◽

Mood Stabilizers ◽

Mitogen Activated Protein Kinase ◽

Facilitatory Effect

One strategy to understand bipolar disorder is to study the mechanism of action of mood-stabilizing drugs, such as valproic acid and lithium. This approach has implicated a number of intracellular signalling elements, such as GSK3β (glycogen synthase kinase 3β), ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) or protein kinase C. However, lamotrigine does not seem to modulate any of these targets, which is intriguing given that its profile in the clinic differs from that of valproic acid or lithium, with greater efficacy to prevent episodes of depression than mania. The primary target of lamotrigine is the voltage-gated sodium channel, but it is unclear why inhibition of these channels might confer antidepressant efficacy. In healthy volunteers, we found that lamotrigine had a facilitatory effect on the BOLD (blood-oxygen-level-dependent) response to TMS (transcranial magnetic stimulation) of the prefrontal cortex. This effect was in contrast with an inhibitory effect of lamotrigine when TMS was applied over the motor cortex. In a follow-up study, a similar prefrontal specific facilitatory effect was observed in a larger cohort of healthy subjects, whereas valproic acid inhibited motor and prefrontal cortical TMS-induced BOLD response. In vitro, we found that lamotrigine (3–10 μM) enhanced the power of gamma frequency network oscillations induced by kainic acid in the rat hippocampus, an effect that was not observed with valproic acid (100 μM). These data suggest that lamotrigine has a positive effect on corticolimbic network function that may differentiate it from other mood stabilizers. The results are also consistent with the notion of corticolimbic network dysfunction in bipolar disorder.

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Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3192 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3192-3203 ◽

Cited By ~ 22

Author(s):

K M Pickham ◽

A N Meyer ◽

J Li ◽

D J Donoghue

Keyword(s):

Protein Kinase ◽

Oocyte Maturation ◽

Xenopus Oocytes ◽

Germinal Vesicle ◽

Cyclin B1 ◽

Phosphorylation Sites ◽

Germinal Vesicle Breakdown ◽

Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

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Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2790727 ◽

1991 ◽

Vol 279 (3) ◽

pp. 727-732 ◽

Cited By ~ 20

Author(s):

G B Sala-Newby ◽

A K Campbell

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Specific Activity ◽

Phosphorylation Site ◽

Live Cells ◽

Phosphorylation Sites ◽

Dependent Protein Kinase ◽

Ph Optimum ◽

Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

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Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5

Journal of Virology ◽

10.1128/jvi.00271-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9195-9205 ◽

Cited By ~ 23

Author(s):

Dipankar Bhattacharya ◽

Mayuri ◽

S. M. Best ◽

R. Perera ◽

R. J. Kuhn ◽

...

Keyword(s):

Protein Kinase ◽

Yellow Fever Virus ◽

Nonstructural Protein ◽

Protein Kinase G ◽

Phosphorylation Sites ◽

Histidine Residue ◽

Phosphorylation Event ◽

Langat Virus ◽

And Function

ABSTRACT Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.

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