Genetic Evidence for Pak1 Autoinhibition and Its Release by Cdc42

ABSTRACT Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.

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Multiple regulatory domains on the Byr2 protein kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5876 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5876-5887 ◽

Cited By ~ 59

Author(s):

H Tu ◽

M Barr ◽

D L Dong ◽

M Wigler

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Intramolecular Interaction ◽

Mitogen Activated Protein Kinase ◽

Regulatory Domain ◽

Regulatory Domains ◽

Kinase Catalytic Domain ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

G Protein Alpha Subunit

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.

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PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system.

The Journal of Cell Biology ◽

10.1083/jcb.128.3.263 ◽

1995 ◽

Vol 128 (3) ◽

pp. 263-271 ◽

Cited By ~ 211

Author(s):

J Staudinger ◽

J Zhou ◽

R Burgess ◽

S J Elledge ◽

E N Olson

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Hybrid System ◽

Catalytic Domain ◽

Yeast Two Hybrid ◽

Kinase C ◽

Yeast Two Hybrid System ◽

Wide Range ◽

Two Hybrid

Protein kinase C (PKC) plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors. Upon activation by diacylglycerol, PKC translocates to different subcellular sites where it phosphorylates numerous proteins, most of which are unidentified. We used the yeast two-hybrid system to identify proteins that interact with activated PKC alpha. Using the catalytic region of PKC fused to the DNA binding domain of yeast GAL4 as "bait" to screen a mouse T cell cDNA library in which cDNA was fused to the GAL4 activation domain, we cloned several novel proteins that interact with C-kinase (PICKs). One of these proteins, designated PICK1, interacts specifically with the catalytic domain of PKC and is an efficient substrate for phosphorylation by PKC in vitro and in vivo. PICK1 is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK1 and other PICKs may play important roles in mediating the actions of PKC.

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Subunit Interactions in the mariner Transposase

Genetics ◽

10.1093/genetics/144.3.1087 ◽

1996 ◽

Vol 144 (3) ◽

pp. 1087-1095 ◽

Cited By ~ 3

Author(s):

Allan R Lohe ◽

David T Sullivan ◽

Daniel L Hartl

Keyword(s):

Hybrid System ◽

Catalytic Domain ◽

Wild Type ◽

Target Element ◽

Yeast Two Hybrid ◽

Subunit Interactions ◽

Hsp70 Promoter ◽

Yeast Two Hybrid System ◽

Transposition Reaction ◽

Two Hybrid

Abstract We have studied the Mos1 transposase encoded by the transposable element mariner. This transposase is a member of the “D,D(35)E” superfamily of proteins exhibiting the motif D,D(34)D. It is not known whether this transposase, or other eukaryote transposases manifesting the D,D(35)E domain, functions in a multimeric form. Evidence for oligomerization was found in the negative complementation of Mos1 by an EMS-induced transposase mutation in the catalytic domain. The transposase produced by this mutation has a glycine-to-arginine replacement at position 292. The G292R mutation strongly interferes with the ability of wild-type transposase to catalyze excision of a target element. Negative complementation was also observed for two other EMS mutations, although the effect was weaker than observed with G292R. Results from the yeast two-hybrid system also imply that Mos1 subunits interact, suggesting the possibility of subunit oligomerization in the transposition reaction. Overproduction of Mos1 subunits through an hsp70 promoter also inhibits excision of the target element, possibly through autoregulatory feedback on transcription or through formation of inactive or less active oligomers. The effects of both negative complementation and overproduction may contribute to the regulation of mariner transposition.

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Interaction of the Repressors Nrg1 and Nrg2 With the Snf1 Protein Kinase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/158.2.563 ◽

2001 ◽

Vol 158 (2) ◽

pp. 563-572 ◽

Cited By ~ 1

Author(s):

Valmik K Vyas ◽

Sergei Kuchin ◽

Marian Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Hybrid System ◽

Fluorescent Protein ◽

Zinc Finger Protein ◽

Glucose Repression ◽

Snf1 Kinase ◽

Cell Extracts ◽

Green Fluorescent ◽

Two Hybrid

Abstract The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C2H2 zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

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Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

Biochemical Journal ◽

10.1042/bj20111536 ◽

2012 ◽

Vol 442 (1) ◽

pp. 105-118 ◽

Cited By ~ 30

Author(s):

Craig R. Pigott ◽

Halina Mikolajek ◽

Claire E. Moore ◽

Stephen J. Finn ◽

Curtis W. Phippen ◽

...

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Functional Organization ◽

Elongation Factor ◽

Kinase Domain ◽

Conserved Residues ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Kinase Catalytic Domain ◽

Eukaryotic Elongation Factor

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

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Effects of inhibitors of protein kinase C and calpain in experimental delayed cerebral vasospasm

Journal of Neurosurgery ◽

10.3171/jns.1992.76.1.0111 ◽

1992 ◽

Vol 76 (1) ◽

pp. 111-118 ◽

Cited By ~ 48

Author(s):

Nobutaka Minami ◽

Eiichi Tani ◽

Yukio Maeda ◽

Ikuya Yamaura ◽

Masahiro Fukami

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Catalytic Domain ◽

Limited Proteolysis ◽

Maximum Effect ◽

Regulatory Domain ◽

Calphostin C ◽

Delayed Cerebral Vasospasm ◽

Kinase C ◽

Basilar Arteries

✓ Vasospasm was produced in adult mongrel dogs by a two-hemorrhage method, and the spastic basilar arteries were exposed via the transclival route on Day 7. Tonic contraction was produced in the normal canine basilar arteries by a local application of KCl or serotonin after transclival exposure. The exposed spastic and tonic basilar arteries then received a topical application of the following: 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7), a potent inhibitor of protein kinase C acting at the catalytic domain; calphostin C, a specific inhibitor of protein kinase C acting at the regulatory domain; or calpeptin, a selective inhibitor of calpain. Both spastic and tonic basilar arteries were effectively dilated by H-7. Calphostin C caused only slight dilation of spastic basilar arteries but moderate dilation of tonic basilar arteries. Dilation in response to calpeptin was remarkable in the spastic basilar arteries but slight in the tonic basilar arteries. The doses of calphostin C and calpeptin required to obtain maximum effect were markedly lower in the tonic model than in the spastic model. The spastic and tonic models had a similar dose-dependent response to H-7 but quite a different response to calphostin C or calpeptin, suggesting a difference in the function of protein kinase C and calpain in the two models. Furthermore, the effect of calphostin C on the reversal of vasospasm was increased significantly after topical treatment with calpeptin. It is suggested that the majority of the catalytic domain of protein kinase C is dissociated from the regulatory domain, probably by a limited proteolysis with calpain, and is markedly activated in vasospasm.

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