Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recently described protein kinase C-related kinase (PRK) family is comprised of at least three members: PRK1, PRK2 and PRK3. Here the expression, purification and characterization of the ubiquitously expressed isoform, PRK1, is described. The enzyme was expressed in COS 7 cells and subsequently purified to apparent homogeneity by sequential column chromatography. The purified PRK1 protein migrates as a single 120 kDa polypeptide on SDS/PAGE. It displays a substrate specificity that in part resembles that of protein kinase C (PKC); however, unlike PKC, it is not activated by any combination of phorbol esters, diacylglycerol and Ca2+. Nevertheless, it can be activated by limited proteolysis, indicating a negative regulatory role for the N-terminal domain(s). PRK1 is also activated by phospholipids. The physiological relevance of this activation is discussed.
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PMID:Expression, purification and characterization of the ubiquitous protein kinase C-related kinase 1. 761 73

A homologue of human protein kinase C (PKC)-related kinase-2, PRK2, which had previously escaped identification in normal mammalian tissues, was isolated from rat liver as the protease-activated kinase (PAK) originally named PAK-2. The 130-kDa cytosolic enzyme was purified to homogeneity and shown by tryptic peptide and reverse transcriptase- polymerase chain reaction (RT-PCR)-amplified rat cDNA sequence analyses to be structurally related to the 116-kDa rat hepatic PAK-1/protein kinase N (PKN) and, even more closely (95% sequence identity) to the 130-kDa human PKC-related kinase, PRK2. Rat myeloma RNA was used as the RT-PCR template because of its relative abundance in PAK-2/PRK2 mRNA compared with liver and other rat tissues. The catalytic properties of PAK-2/PRK2 in many respects resembled those of hepatic PAK-1/PKN, but were distinguished by more favorable kinetics with several peptide substrates, and greater sensitivity to PKC pseudosubstrate and polybasic amino acid inhibitors. PAK-2/PRK2 was also activated by lipids, particularly cardiolipin and to a lesser extent by other acidic phospholipids and unsaturated fatty acids. Cardiolipin activation was most evident with autophosphorylation and histone H2B phosphorylation, but only marginally evident with the favored ribosomal S6-(229-239) peptide substrate for the protease-activated kinase activity. It was concluded that PAK-2 is the rat homologue of human PRK2, with biochemical properties distinct from although overlapping those of the PAK-1/PKN/PRK1 isoform.
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PMID:Isolation and characterization of a structural homologue of human PRK2 from rat liver. Distinguishing substrate and lipid activator specificities. 909 45

PKN is a fatty acid- and Rho GTPase-activated protein kinase whose catalytic domain in the carboxyl terminus is homologous to those of protein kinase C (PKC) family members. The amino terminal region of PKN is suggested to function as a regulatory domain, since tryptic cleavage or the binding of Rho GTPase to this region results in protein kinase activation of PKN. The structural basis for the regulation of PKN was investigated by analyzing the activity of a series of deletion/site-directed mutants expressed in insect cells. The amino-terminally truncated form of PKN (residue 455-942) showed low basal activity similar to that of the wild-type enzyme, and was arachidonic acid-dependent. However, further deletion (residue 511-942) resulted in a marked increase in the basal activity and a decrease in the arachidonic acid dependency. A (His)(6)-tagged protein comprising residues 455-511 of PKN (designated His-Ialpha) inhibited the kinase activity of the catalytic fragment of PKN in a concentration-dependent manner in competition with substrate (K(i) = 0.6+/-0.2 microM). His-Ialpha also inhibited the activity of the catalytic fragment of PRK2, an isoform of PKN, but had no inhibitory effect on protein kinase A or protein kinase Cdelta. The IC(50) value obtained in the presence of 40 microM arachidonic acid was two orders of magnitude greater than that in the absence of the modifier. These results indicate that this protein fragment functions as a specific inhibitor of PKN and PRK2, and that arachidonic acid relieves the catalytic activity of wild-type PKN from autoinhibition by residues 455-511 of PKN. Autophosphorylation of wild-type PKN increased the protein kinase activity, however, substitution of Thr64, Ser374, or Thr531 in the regulatory region of PKN with alanine, abolished this effect. Substitution of Thr774 in the activation loop of the catalytic domain of PKN with alanine completely abolished the protein kinase activity. These results suggest that these phosphorylation sites are also important in the regulation of the PKN kinase activity. Potential differences in the mechanism of activation between the catalytic regions of PKN and PRK2 are also discussed.
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PMID:Mutational analysis of the regulatory mechanism of PKN: the regulatory region of PKN contains an arachidonic acid-sensitive autoinhibitory domain. 1046 62

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.
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PMID:A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1. 1076 42

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c-fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.
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PMID:The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element. 1084 71

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.
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PMID:Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1. 1100 71

The multi-site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T-loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1). In this study, we demonstrate that in embryonic stem (ES) cells lacking PDK1 (PDK1-/- cells), the intracellular levels of endogenously expressed PKCalpha, PKCbetaI, PKCgamma, PKCdelta, PKCepsilon, and PKC-related kinase-1 (PRK1) are vastly reduced compared to control ES cells (PDK1+/+ cells). The levels of PKCzeta and PRK2 protein are only moderately reduced in the PDK1-/- ES cells. We demonstrate that in contrast to PKCzeta expressed PDK1+/+ ES cells, PKCzeta in ES cells lacking PDK1 is not phosphorylated at its T-loop residue. This provides the first genetic evidence that PKCzeta is a physiological substrate for PDK1. In contrast, PRK2 is still partially phosphorylated at its T-loop in PDK1-/- cells, indicating the existence of a PDK1-independent mechanism for the phosphorylation of PRK2 at this residue.
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PMID:Further evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms. 1107 82

The mechanism by which PDK1 regulates AGC kinases remains unclear. To further understand this process, we performed a yeast two-hybrid screen using PDK1 as bait. PKC-zeta, PKC-delta, and PRK2 were identified as interactors of PDK1. A combination of yeast two-hybrid binding assays and coprecipitation from mammalian cells was used to characterize the nature of the PDK1-PKC interaction. The presence of the PH domain of PDK1 inhibited the interaction of PDK1 with the PKCs. A contact region of PDK1 was mapped between residues 314 and 408. The interaction of PDK1 with the PKCs required the full-length PKC-zeta and -delta proteins apart from their C-terminal tails. PDK1 was able to phosphorylate full-length PKC-zeta and -delta but not PKC-zeta and -delta constructs containing the PDK1 phosphorylation site but lacking the C-terminal tails. A C-terminal PRK2 fragment, normally produced by caspase-3 cleavage during apoptosis, inhibited PDK1 autophosphorylation by >90%. The ability of PDK1 to phosphorylate PKC-zeta and -delta in vitro was also markedly inhibited by the PRK2 fragment. Additionally, generation of the PRK2 fragment in vivo inhibited by >90% the phosphorylation of endogenous PKC-zeta by PDK1. In conclusion, these results show that the C-terminal tail of PKC is a critical determinant for PKC-zeta and -delta phosphorylation by PDK1. Moreover, the C-terminal PRK2 fragment acts as a potent negative regulator of PDK1 autophosphorylation and PDK1 kinase activity against PKC-zeta and -delta. As the C-terminal PRK2 fragment is naturally generated during apoptosis, this may provide a mechanism of restraining prosurvival signals during apoptosis.
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PMID:Regulation of both PDK1 and the phosphorylation of PKC-zeta and -delta by a C-terminal PRK2 fragment. 1178 Oct 95

PKN is a serine/threonine protein kinase that has a catalytic domain homologous to protein kinase C (PKC) family members and a unique regulatory region containing antiparallel coiled-coil (ACC) domains. PKN is the first identified serine/threonine protein kinase that can bind to and be activated by a small GTPase Rho, and it can also be activated by fatty acids such as arachidonic acid in vitro. PKN is widely distributed in various organisms such as mammal, frog, fly, and starfish. There are at least three different isoforms of PKN (PKNalpha/PAK-1/PRK-1, PKNbeta, and PRK2/PAK-2/PKNgamma) in mammals, each of which shows different enzymological properties, tissue distribution, and varied functions.
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PMID:The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC. 1276 Nov 94

PKN is a serine/threonine protein kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC) in the carboxyl-terminal region and three repeats of the antiparallel coiled coil (ACC) domain in the amino-terminal region. Mammalian PKN has three isoforms each derived from different genes, PKN1 (PKNalpha/PRK1/PAK1), PKN2 (PRK2/PAK2/PKNgamma), and PKN3 (PKNbeta). PKN isoforms show different enzymatic properties and tissue distributions and have been implicated in various distinct cellular processes (reviewed in Mukai [2003]). This chapter discusses methods to prepare purified enzymes and to assay substrate phosphorylation activities.
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PMID:Purification and kinase assay of PKN. 1647 61


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