Substrate Targeting of the Yeast Cyclin-Dependent Kinase Pho85p by the Cyclin Pcl10p

ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) catalytic subunit with multiple regulatory roles thought to be specified by association with different cyclin partners (Pcls). Pcl10p is one of four Pcls with little sequence similarity to cyclins involved in cell cycle control. It has been implicated in specifying the phosphorylation of glycogen synthase (Gsy2p). We report that recombinant Pho85p and Pcl10p produced inEscherichia coli reconstitute an active Gsy2p kinase in vitro. Gsy2p phosphorylation required Pcl10p, occurred at physiologically relevant sites, and resulted in inactivation of Gsy2p. The activity of the reconstituted enzyme was even greater than Pho85p-Pcl10p isolated from yeast, and we conclude that, unlike many Cdks, Pho85p does not require phosphorylation for activity. Pcl10p formed complexes with Gsy2p, as judged by (i) gel filtration of recombinant Pcl10p and Gsy2p, (ii) coimmunoprecipitation from yeast cell lysates, and (iii) enzyme kinetic behavior consistent with Pcl10p binding the substrate. Synthetic peptides modeled on the sequences of known Pho85p sites were poor substrates with highKm values, and we propose that Pcl10p-Gsy2p interaction is important for substrate selection. Gel filtration of yeast cell lysates demonstrated that most Pho85p was present as a monomer, although a portion coeluted in high-molecular-weight fractions with Pcl10p and Gsy2p. Overexpression of Pcl10p sequestered most of the Pho85p into association with Pcl10p. We suggest a model for Pho85p function in the cell whereby cyclins like Pcl10p recruit Pho85p from a pool of monomers, both activating the kinase and targeting it to substrate.

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Proteomic and biochemical comparison of the cellular interaction partners of human VPS33A and VPS33B

10.1101/236695 ◽

2017 ◽

Author(s):

Morag R. Hunter ◽

Geoffrey G. Hesketh ◽

Anne-Claude Gingras ◽

Stephen C. Graham

Keyword(s):

Sequence Similarity ◽

Domain Architecture ◽

Gel Filtration ◽

Cellular Interaction ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Protein Subunit ◽

Over Expression ◽

Biochemical Comparison

ABSTRACTMulti-subunit tethering complexes control membrane fusion events in eukaryotic cells. CORVET and HOPS are two such multi-subunit tethering complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named ‘CHEVI’), analogous in configuration to CORVET and HOPS. We utilised the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting these proteins have distinct sub-cellular localisations. Consistent with previous reports, we observed that VPS33A was co-localised with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although in this assay VPS33A clearly co-localised with several subunits of CORVET and HOPS, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localise with VPS33B. Instead, we identified two novel VPS33B-interacting proteins, VPS53 and CCDC22. CCDC22 co-immunoprecipitated with VPS33B and VIPAR in over-expression conditions and interacts directly with the VPS33B-VIPAR complex in vitro. However, CCDC22 does not appear to co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates. We also observed that the protein complex in HEK293T cells which contained VPS33B and VIPAR was considerably smaller than CORVET/HOPS, suggesting that, unlike VPS33A, VPS33B does not assemble into a large stable multi-subunit tethering complex.

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Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4694 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4694-4705 ◽

Cited By ~ 79

Author(s):

S J Baker ◽

T K Kerppola ◽

D Luk ◽

M T Vandenberg ◽

D R Marshak ◽

...

Keyword(s):

Protein Kinase ◽

Dna Binding ◽

Glycogen Synthase ◽

In Vitro Transcription ◽

Binding Activity ◽

Dna Binding Activity ◽

Serum Stimulation ◽

Dependent Protein

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

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Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2

Biochemical Journal ◽

10.1042/bj3390319 ◽

1999 ◽

Vol 339 (2) ◽

pp. 319-328 ◽

Cited By ~ 265

Author(s):

Takayasu KOBAYASHI ◽

Philip COHEN

Keyword(s):

Hydrogen Peroxide ◽

Protein Kinase ◽

Catalytic Domain ◽

Glycogen Synthase ◽

Pleckstrin Homology Domain ◽

Dependent Protein Kinase ◽

293 Cells ◽

Dependent Protein ◽

Dependent Pathway

The PtdIns(3,4,5)P3-dependent activation of protein kinase B (PKB) by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2 respectively) is a key event in mediating the effects of signals that activate PtdIns 3-kinase. The catalytic domain of serum- and glucocorticoid-regulated protein kinase (SGK) is 54% identical with that of PKB and, although lacking the PtdIns(3,4,5)P3-binding pleckstrin-homology domain, SGK retains the residues that are phosphorylated by PDK1 and PDK2, which are Thr256 and Ser422 in SGK. Here we show that PDK1 activates SGK in vitro by phosphorylating Thr256. We also show that, in response to insulin-like growth factor-1 (IGF-1) or hydrogen peroxide, transfected SGK is activated in 293 cells via a PtdIns 3-kinase-dependent pathway that involves the phosphorylation of Thr256 and Ser422. The activation of SGK by PDK1 in vitro is unaffected by PtdIns(3,4,5)P3, abolished by the mutation of Ser422 to Ala, and greatly potentiated by mutation of Ser422 to Asp (although this mutation does not activate SGK itself). Consistent with these findings, the Ser422Asp mutant of SGK is activated by phosphorylation (probably at Thr256) in unstimulated 293 cells, and activation is unaffected by inhibitors of PtdIns 3-kinase. Our results are consistent with a model in which activation of SGK by IGF-1 or hydrogen peroxide is initiated by a PtdIns(3,4,5)P3-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the PtdIns(3,4,5)P3-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1. Like PKB, SGK preferentially phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs, and SGK and PKB inactivate glycogen synthase kinase-3 similarly in vitroand in co-transfection experiments. These findings raise the possibility that some physiological roles ascribed to PKB on the basis of the overexpression of constitutively active PKB mutants might be mediated by SGK.

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cAMP-Dependent Activation of Mammalian Target of Rapamycin (mTOR) in Thyroid Cells. Implication in Mitogenesis and Activation of CDK4

Molecular Endocrinology ◽

10.1210/me.2010-0087 ◽

2010 ◽

Vol 24 (7) ◽

pp. 1453-1468 ◽

Cited By ~ 43

Author(s):

Sara Blancquaert ◽

Lifu Wang ◽

Sabine Paternot ◽

Katia Coulonval ◽

Jacques E. Dumont ◽

...

Keyword(s):

Protein Kinase ◽

Glycogen Synthase ◽

Mammalian Target Of Rapamycin ◽

Cyclin D3 ◽

Target Of Rapamycin ◽

Thyroid Cells ◽

Dependent Protein ◽

Rat Thyroid ◽

Amp Activated Protein Kinase

Abstract How cAMP-dependent protein kinases [protein kinase A (PKA)] transduce the mitogenic stimulus elicited by TSH in thyroid cells to late activation of cyclin D3-cyclin-dependent kinase 4 (CDK4) remains enigmatic. Here we show in PC Cl3 rat thyroid cells that TSH/cAMP, like insulin, activates the mammalian target of rapamycin (mTOR)-raptor complex (mTORC1) leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMP-activated protein kinase and glycogen synthase kinase 3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate protein kinase B or induce tuberous sclerosis complex 2 phosphorylation at T1462 and Y1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity that was associated with augmented 4E-BP1 binding to raptor. This could be caused in part by T246 phosphorylation of PRAS40, which was found as an in vitro substrate of PKA. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and retinoblastoma protein phosphorylation induced by TSH and insulin. Although rapamycin reduced cyclin D3 accumulation, the abundance of cyclin D3-CDK4 complexes was not affected. However, rapamycin inhibited the activity of these complexes by decreasing the TSH and insulin-mediated stimulation of activating T172 phosphorylation of CDK4. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 T172-phosphorylation.

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Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper

Biochemical Journal ◽

10.1042/bj3260853 ◽

1997 ◽

Vol 326 (3) ◽

pp. 853-859 ◽

Cited By ~ 46

Author(s):

Sergio LIZANO ◽

Bruno LOMONTE ◽

Jay W. FOX ◽

José Maréa GUTIÉRREZ

Keyword(s):

Phospholipase A2 ◽

Molecular Mass ◽

Biochemical Characterization ◽

Biological Activities ◽

Sequence Similarity ◽

Gel Filtration ◽

Cytolytic Activity ◽

Bothrops Asper

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23–25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23–25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I–IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

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Function of Mammalian LKB1 and Ca2+/Calmodulin-dependent Protein Kinase Kinase α as Snf1-activating Kinases in Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m501887200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 21804-21809 ◽

Cited By ~ 84

Author(s):

Seung-Pyo Hong ◽

Milica Momcilovic ◽

Marian Carlson

Keyword(s):

Catalytic Activity ◽

Protein Kinase ◽

Mammalian Cells ◽

Metabolic Regulation ◽

Sequence Similarity ◽

Snf1 Kinase ◽

Glucose Signaling ◽

Kinase Cascade ◽

Dependent Protein

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation in response to stress. In the yeast Saccharomyces cerevisiae, the Snf1 kinase cascade comprises three Snf1-activating kinases, Pak1, Tos3, and Elm1. The only established mammalian AMPK kinase is LKB1. We show that LKB1 functions heterologously in yeast. In pak1Δ tos3Δ elm1Δ cells, LKB1 activated Snf1 catalytic activity and conferred a Snf+ growth phenotype. Coexpression of STRADα and MO25α, which form a complex with LKB1, enhanced LKB1 function. Thus, the Snf1/AMPK kinase cascade is functionally conserved between yeast and mammals. Ca2+/calmodulin-dependent kinase kinase (CaMKK) shows more sequence similarity to Pak1, Tos3, and Elm1 than does LKB1. When expressed in pak1Δ tos3Δ elm1Δ cells, CaMKKα activated Snf1 catalytic activity, restored the Snf+ phenotype, and also phosphorylated the activation loop threonine of Snf1 in vitro. These findings indicate that CaMKKα is a functional member of the Snf1/AMPK kinase family and support CaMKKα as a likely candidate for an AMPK kinase in mammalian cells. Analysis of the function of these heterologous kinases in yeast provided insight into the regulation of Snf1. When activated by LKB1 or CaMKKα, Snf1 activity was significantly inhibited by glucose, suggesting that a mechanism independent of the activating kinases can mediate glucose signaling in yeast. Finally, this analysis provided evidence that Pak1 functions in another capacity, besides activating Snf1, to regulate the nuclear enrichment of Snf1 protein kinase in response to carbon stress.

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GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease

Journal of Clinical Medicine ◽

10.3390/jcm8101751 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1751 ◽

Cited By ~ 1

Author(s):

Ko ◽

Chiou ◽

Wong ◽

Wang ◽

Lai ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Protein Kinase ◽

Glycogen Synthase ◽

Tau Phosphorylation ◽

In Vitro Assays ◽

Anchoring Proteins ◽

Dependent Protein ◽

Induced Pluripotent ◽

Isogenic Mutants

Based on the protein kinase A (PKA)/GSK3β interaction protein (GSKIP)/glycogen synthase kinase 3β (GSK3β) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3β-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3β with PKA but not Ca2+/calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer’s disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA–Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein (APPWT/D678H) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3β, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3β function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis.

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Anticoagulant Activities of Lung and Mucosal Heparin

10.1055/s-0039-1680371 ◽

1977 ◽

Author(s):

T. W. Barrowcliffe ◽

E. A. Johnson ◽

C. A. Eggleton ◽

D. P. Thomas

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Gel Filtration ◽

High Ratio ◽

Low Molecular Weight ◽

Multiple Role ◽

Heparin Activity ◽

Molecular Weight Fractions

Interaction with antithrombin III is thought to be the main mechanism whereby heparin exerts an anticoagulant effect, but measurements of this specific heparin activity by an anti-Xa assay do not always agree with measurements made by ‘multiple role’ assays, such as APTT or pharmacopoeial assays. Two batches of lung heparin had APTT activities in vitro about 1.4 times those found by anti-Xa, whereas in several batches of porcine mucosal heparin this ratio was about 0.8. All assays by both methods were carried out against the 3rd International Standard for heparin. After gel filtration, lung heparin maintained a high ratio of APTT to anti-Xa activity in all except the low molecular weight fractions, where the two activities were both about 60 i. u./mg. In contrast, low molecular weight mucosal fractions had negligible APTT activity, but high (120 i. u./mg) activity by anti-Xa assay. A nominal 1000 units of lung heparin injected I. V. into volunteers gave peak anti-Xa levels of about 0.2 i. u./ml; a comparable injection of mucosal heparin gave peak levels of about 0.3 i. u./ml. The resulting ratio agreed with the anti-Xa activities of these two batches in vitro. However, in vivo, APTT levels with both heparins were less than half the anti-Xa levels, and 50 mins. after injection there was virtually no effect on the APTT, while heparin levels by anti-Xa remained about 0.1 i. u./ml. Although their APTT activities were comparable, lung heparin had much less anti-Xa potentiating effect than mucosal heparin, both in vitro and in vivo; this has important implications for the assay and clinical use of heparin.

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Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4694-4705.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4694-4705

Author(s):

S J Baker ◽

T K Kerppola ◽

D Luk ◽

M T Vandenberg ◽

D R Marshak ◽

...

Keyword(s):

Protein Kinase ◽

Dna Binding ◽

Glycogen Synthase ◽

In Vitro Transcription ◽

Binding Activity ◽

Dna Binding Activity ◽

Serum Stimulation ◽

Dependent Protein

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Purification of the Antihemophilic Factor by Gel Filtration on Agarose

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651394 ◽

1969 ◽

Vol 22 (03) ◽

pp. 577-583 ◽

Cited By ~ 3

Author(s):

M.M.P Paulssen ◽

A.C.M.G.B Wouterlood ◽

H.L.M.A Scheffers

Keyword(s):

Factor Viii ◽

Plasma Proteins ◽

Large Scale ◽

Gel Filtration ◽

Large Scale Preparation ◽

Scale Preparation ◽

Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

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