scholarly journals AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

1995 ◽  
Vol 15 (4) ◽  
pp. 2304-2310 ◽  
Author(s):  
K Datta ◽  
T F Franke ◽  
T O Chan ◽  
A Makris ◽  
S I Yang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Protein Complexes ◽  
Ph Domain ◽  
Terminal Portion ◽  
Protein Protein Interaction ◽  
Kinase C ◽  
Nih 3T3

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

scholarly journals Respiratory-induced coenzyme Q biosynthesis is regulated by a phosphorylation cycle of Cat5p/Coq7p

2011 ◽  
Vol 440 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Alejandro Martín-Montalvo ◽  
Isabel González-Mariscal ◽  
Sergio Padilla ◽  
Manuel Ballesteros ◽  
David L. Brautigan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Regulatory Mechanism ◽  
Coenzyme Q ◽  
In Vitro Assays ◽  
Respiratory Metabolism ◽  
Total Absence ◽  
Kinase C ◽  
Pkc Protein Kinase C

CoQ6 (coenzyme Q6) biosynthesis in yeast is a well-regulated process that requires the final conversion of the late intermediate DMQ6 (demethoxy-CoQ6) into CoQ6 in order to support respiratory metabolism in yeast. The gene CAT5/COQ7 encodes the Cat5/Coq7 protein that catalyses the hydroxylation step of DMQ6 conversion into CoQ6. In the present study, we demonstrated that yeast Coq7 recombinant protein purified in bacteria can be phosphorylated in vitro using commercial PKA (protein kinase A) or PKC (protein kinase C) at the predicted amino acids Ser20, Ser28 and Thr32. The total absence of phosphorylation in a Coq7p version containing alanine instead of these phospho-amino acids, the high extent of phosphorylation produced and the saturated conditions maintained in the phosphorylation assay indicate that probably no other putative amino acids are phosphorylated in Coq7p. Results from in vitro assays have been corroborated using phosphorylation assays performed in purified mitochondria without external or commercial kinases. Coq7p remains phosphorylated in fermentative conditions and becomes dephosphorylated when respiratory metabolism is induced. The substitution of phosphorylated residues to alanine dramatically increases CoQ6 levels (256%). Conversely, substitution with negatively charged residues decreases CoQ6 content (57%). These modifications produced in Coq7p also alter the ratio between DMQ6 and CoQ6 itself, indicating that the Coq7p phosphorylation state is a regulatory mechanism for CoQ6 synthesis.

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

1995 ◽  
Vol 128 (3) ◽  
pp. 263-271 ◽  
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.

scholarly journals The regulatory domain of protein kinase C-ε restricts the catalytic-domain-specificity

1991 ◽  
Vol 276 (1) ◽  
pp. 257-260 ◽  
Author(s):  
C Pears ◽  
D Schaap ◽  
P J Parker
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Structural Basis ◽  
Regulatory Domain ◽  
Substrate Selection ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Protein kinase C (PKC) consists of a family of closely related enzymes that can be divided into two subfamilies (alpha, beta and gamma and delta, epsilon and zeta) on the basis of primary sequence. Functional differences have also been described; thus PKC-alpha, PKC-beta and PKC-gamma readily phosphorylate histone IIIS in vitro, whereas PKC-epsilon will not employ this substrate efficiently. We have previously demonstrated, however, that proteolytic cleavage of PKC-epsilon generates a constitutive kinase activity that is an efficient histone IIIS kinase [Schaap, Hsuan, Totty & Parker (1990) Eur. J. Biochem. 191, 431-435]. In order to investigate the structural basis for this switch in specificity, we have constructed a chimaeric protein containing the regulatory domain of PKC-epsilon fused to the catalytic domain of PKC-gamma. When this is expressed in COS1 cells the chimaeric kinase shows a substrate-specificity similar to that of PKC-epsilon rather than to that of PKC-gamma. This demonstrates a role for the regulatory domain in substrate selection of PKC-epsilon.

scholarly journals Human Cytomegalovirus Protein Kinase UL97 Forms a Complex with the Tegument Phosphoprotein pp65

2007 ◽  
Vol 81 (19) ◽  
pp. 10659-10668 ◽  
Author(s):  
Jeremy P. Kamil ◽  
Donald M. Coen
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Opportunistic Infections ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Immunocompromised Host ◽  
Protein Protein Interaction ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACT UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.

scholarly journals Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

1997 ◽  
Vol 17 (2) ◽  
pp. 732-741 ◽  
Author(s):  
H Cai ◽  
U Smola ◽  
V Wixler ◽  
I Eisenmann-Tappe ◽  
M T Diaz-Meco ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dominant Negative ◽  
Inhibitory Effects ◽  
Raf Kinase ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha ◽  
Nih 3T3

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.

Regulation of novel protein kinase C ε by phosphorylation

2002 ◽  
Vol 363 (3) ◽  
pp. 537-545 ◽  
Author(s):  
Vittoria CENNI ◽  
Heike DÖPPLER ◽  
Erica D. SONNENBURG ◽  
Nadir MARALDI ◽  
Alexandra C. NEWTON ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
Kinase Domain ◽  
Activation Loop ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Hydrophobic Motif ◽  
Nih 3T3

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKC∊ isoform, these are Thr566 in the activation loop, Thr710 in the turn motif and Ser729 in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKC∊ kinase activity in controlling the phosphorylation of Thr566 and Ser729. In NIH 3T3 fibroblasts, PKC∊ migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr566 and Ser729. Cells transfected with an active PDK-1 allele also resulted in increased PKC∊ Thr566 and Ser729 phosphorylation, whereas an active myristoylated PKC∊ mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKC∊ were not phosphorylated at Ser729 in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr566, an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKC∊ in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P3, concomitant with an increase in Ser729 phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

scholarly journals Molecular cloning of a second form of rac protein kinase.

1991 ◽  
Vol 2 (12) ◽  
pp. 1001-1009 ◽  
Author(s):  
P F Jones ◽  
T Jakubowicz ◽  
B A Hemmings
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Cell Lines ◽  
Catalytic Domain ◽  
Cell Types ◽  
In Vitro Translation ◽  
Cdna Libraries ◽  
Beta Form

A novel serine/threonine protein kinase (termed rac-PK) has recently been identified and cloned from cDNA libraries derived from the human cell lines MCF-7 and WI38. A second form of this protein kinase, termed rac protein kinase beta, has been identified from cDNAs derived from the same cell lines. These two closely related forms show 90% homology, although the beta form with a predicted Mr 60,200 has a carboxyl terminal extension of 40 amino acids in comparison to the alpha form. This extension has a high serine content with 11 serine residues in the last 30 amino acids. The beta form of the protein has been shown by both in vitro translation and bacterial expression to be approximately 5000 Da larger than the alpha form. rac protein kinase beta is encoded by a 3.4-kb transcript and the alpha form is encoded by a 3.2-kb mRNA. Using gene-specific probes both transcripts were detected in all cell types analyzed, although levels of expression were different for the two forms. The catalytic domain of rac protein kinase beta shows a high degree of homology to both the protein kinase C and cyclic AMP-dependent protein kinase families, and hence rac protein kinases appear to represent a new subfamily of the second messenger serine/threonine protein kinases.

In vitro and in vivo suppression of growth of rat liver epithelial tumor cells by antisense oligonucleotide against protein kinase C-alpha

2000 ◽  
Vol 33 (4) ◽  
pp. 601-608 ◽  
Author(s):  
Shwu-Bin Lin ◽  
Li-Ching Wu ◽  
Siao-Ling Huang ◽  
Hui-Lun Hsu ◽  
Sung-Hwa Hsieh ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tumor Cells ◽  
Rat Liver ◽  
Antisense Oligonucleotide ◽  
Epithelial Tumor ◽  
Kinase C ◽  
Epithelial Tumor Cells
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