PKC-related serine/threonine-protein kinase involved in various processes such as regulation of the intermediate filaments of the actin cytoskeleton, cell migration, tumor cell invasion and transcription regulation. Regulates the cytoskeletal network by phosphorylating proteins such as VIM and neurofilament proteins NEFH, NEFL and NEFM, leading to inhibit their polymerization. Phosphorylates 'Ser-575', 'Ser-637' and 'Ser-669' of MAPT/Tau, lowering its ability to bind to microtubules, resulting in disruption of tubulin assembly. Acts as a key coactivator of androgen receptor (ANDR)-dependent transcription, by being recruited to ANDR target genes and specifically mediating phosphorylation of 'Thr-11' of histone H3 (H3T11ph), a specific tag for epigenetic transcriptional activation that promotes demethylation of histone H3 'Lys-9' (H3K9me) by KDM4C/JMJD2C. Phosphorylates HDAC5, HDAC7 and HDAC9, leading to impair their import in the nucleus. Phosphorylates 'Thr-38' of PPP1R14A, 'Ser-159', 'Ser-163' and 'Ser-170' of MARCKS, and GFAP. Able to phosphorylate RPS6 in vitro.
The 80kDa Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is a major in vivo substrate of protein kinase C (PKC). Here we report that MARCKS is a major substrate for the lipid-activated PKC-related kinase (PRK1) in cell extracts. Furthermore, PRK1 is shown to phosphorylate MARCKS on the same sites as PKC in vitro. Thus, control of MARCKS phosphorylation on these previously identified 'PKC' sites may be regulated under certain circumstances by PRK as well as PKC mediated signalling pathways. The implications for MARCKS as a marker of PKC activation and as a point of signal convergence are discussed.
In addition to the classical activation by ligands, nuclear receptor activity is also regulated by ligand-independent signalling. Here, we unravel a novel signal transduction pathway that links the RhoA effector protein kinase C-related kinase PRK1 to the transcriptional activation of the androgen receptor (AR). Stimulation of the PRK signalling cascade results in a ligand-dependent superactivation of AR. We show that AR and PRK1 interact both in vivo and in vitro. The transactivation unit 5 (TAU-5) located in the N-terminus of AR suffices for activation by PRK1. Thus, TAU-5 defines a novel, signal-inducible transactivation domain. Furthermore, PRK1 promotes a functional complex of AR with the co-activator TIF-2. Importantly, PRK signalling also stimulates AR activity in the presence of adrenal androgens, which are still present in prostate tumour patients subjected to testicular androgen ablation therapy. Moreover, PRK1 activates AR even in the presence of the AR antagonist cyproterone acetate that is used in the clinical management of prostate cancer. Since prostate tumours strongly overexpress PRK1, our data support a model in which AR activity is controlled by PRK signalling.
Rho GTPases regulate multiple signal transduction pathways that influence many aspects of cell behaviour, including migration, morphology, polarity and cell cycle. Through their ability to control the assembly and organization of the actin and microtubule cytoskeletons, Rho and Cdc42 make several key contributions during the mitotic phase of the cell cycle, including spindle assembly, spindle positioning, cleavage furrow contraction and abscission. We now report that PRK2/PKN2, a Ser/Thr kinase and Rho/Rac effector protein, is an essential regulator of both entry into mitosis and exit from cytokinesis in HeLa S3 cells. PRK2 is required for abscission of the midbody at the end of the cell division cycle and for phosphorylation and activation of Cdc25B, the phosphatase required for activation of mitotic cyclin/Cdk1 complexes at the G2/M transition. This reveals an additional step in the mammalian cell cycle controlled by Rho GTPases.
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
PKN is a serine/threonine protein kinase with a catalytic domain homologous to the protein kinase C family and unique N-terminal leucine zipper-like sequences. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that the regulatory domain of PKN interacted with vimentin. We then examined whether PKN would phosphorylate vimentin in vitro. Vimentin proved to be an excellent substrate for PKN, and the phosphorylation of vimentin by PKN occurred in the head domain with the result of a nearly complete inhibition of its filament formation in vitro. Similar results were also obtained with another type III intermediate filament protein, glial fibrillary acidic protein (GFAP). These results raise the possibility that PKN may regulate filament structures of vimentin and GFAP by domain-specific phosphorylation.
Class IIa histone deacetylases (HDACs) -4, -5, -7 and -9 undergo signal-dependent nuclear export upon phosphorylation of conserved serine residues that are targets for 14-3-3 binding. Little is known of other mechanisms for regulating the subcellular distribution of class IIa HDACs. Using a biochemical purification strategy, we identified protein kinase C-related kinase-2 (PRK2) as an HDAC5-interacting protein. PRK2 and the related kinase, PRK1, phosphorylate HDAC5 at a threonine residue (Thr-292) positioned within the nuclear localization signal (NLS) of the protein. HDAC7 and HDAC9 contain analogous sites that are phosphorylated by PRK, while HDAC4 harbors a non-phosphorylatable alanine residue at this position. We provide evidence to suggest that the unique phospho-acceptor cooperates with the 14-3-3 target sites to impair HDAC nuclear import.
The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.
J. Biol. Chem. 271, 9816-9822 (1996)[PubMed:8621664]
PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.
For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
In addition to the classical activation by ligands, nuclear receptor activity is also regulated by ligand-independent signalling. Here, we unravel a novel signal transduction pathway that links the RhoA effector protein kinase C-related kinase PRK1 to the transcriptional activation of the androgen receptor (AR). Stimulation of the PRK signalling cascade results in a ligand-dependent superactivation of AR. We show that AR and PRK1 interact both in vivo and in vitro. The transactivation unit 5 (TAU-5) located in the N-terminus of AR suffices for activation by PRK1. Thus, TAU-5 defines a novel, signal-inducible transactivation domain. Furthermore, PRK1 promotes a functional complex of AR with the co-activator TIF-2. Importantly, PRK signalling also stimulates AR activity in the presence of adrenal androgens, which are still present in prostate tumour patients subjected to testicular androgen ablation therapy. Moreover, PRK1 activates AR even in the presence of the AR antagonist cyproterone acetate that is used in the clinical management of prostate cancer. Since prostate tumours strongly overexpress PRK1, our data support a model in which AR activity is controlled by PRK signalling.
Interacting selectively and non-covalently with chromatin, the network of fibers of DNA, protein, and sometimes RNA, that make up the chromosomes of the eukaryotic nucleus during interphase.
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
J. Biol. Chem. 273, 4811-4814 (1998)[PubMed:9478917]
RhoB has been shown to be an endosomal GTPase both by immunocytochemistry and electron microscopy, however, its role in endocytosis is unknown. Elucidation of the cellular roles of other members of this superfamily of signaling proteins has come with the identification of their downstream partners. We show here that the recently isolated serine/threonine kinase PRK1 is targeted to the endosomal compartment by RhoB. This is established both through immunofluorescence and cell fractionation. PRK1 is shown to interact with activated RhoB in cells and is localized to endosomes through its Rho-binding HR1 domain. Translocation of PRK1 to the endosomal compartment by RhoB is accompanied by a shift in the electrophoretic mobility of the kinase indicative of an accompanying activation.
The small G protein Rho has emerged as a key regulator of cellular events involving cytoskeletal reorganization. Here we report the 2.2 A crystal structure of RhoA bound to an effector domain of protein kinase PKN/PRK1. The structure reveals the antiparallel coiled-coil finger (ACC finger) fold of the effector domain that binds to the Rho specificity-determining regions containing switch I, beta strands B2 and B3, and the C-terminal alpha helix A5, predominantly by specific hydrogen bonds. The ACC finger fold is distinct from those for other small G proteins and provides evidence for the diverse ways of effector recognition. Sequence analysis based on the structure suggests that the ACC finger fold is widespread in Rho effector proteins.
Interacting selectively and non-covalently with a histone, any of a group of water-soluble proteins found in association with the DNA of plant and animal chromosomes. They are involved in the condensation and coiling of chromosomes during cell division and have also been implicated in nonspecific suppression of gene activity.
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
Class IIa histone deacetylases (HDACs) -4, -5, -7 and -9 undergo signal-dependent nuclear export upon phosphorylation of conserved serine residues that are targets for 14-3-3 binding. Little is known of other mechanisms for regulating the subcellular distribution of class IIa HDACs. Using a biochemical purification strategy, we identified protein kinase C-related kinase-2 (PRK2) as an HDAC5-interacting protein. PRK2 and the related kinase, PRK1, phosphorylate HDAC5 at a threonine residue (Thr-292) positioned within the nuclear localization signal (NLS) of the protein. HDAC7 and HDAC9 contain analogous sites that are phosphorylated by PRK, while HDAC4 harbors a non-phosphorylatable alanine residue at this position. We provide evidence to suggest that the unique phospho-acceptor cooperates with the 14-3-3 target sites to impair HDAC nuclear import.
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
The function of a transcription cofactor that activates transcription in conjuction with a ligand-dependent nuclear receptor from a RNA polymerase II promoter; does not bind DNA itself.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
In addition to the classical activation by ligands, nuclear receptor activity is also regulated by ligand-independent signalling. Here, we unravel a novel signal transduction pathway that links the RhoA effector protein kinase C-related kinase PRK1 to the transcriptional activation of the androgen receptor (AR). Stimulation of the PRK signalling cascade results in a ligand-dependent superactivation of AR. We show that AR and PRK1 interact both in vivo and in vitro. The transactivation unit 5 (TAU-5) located in the N-terminus of AR suffices for activation by PRK1. Thus, TAU-5 defines a novel, signal-inducible transactivation domain. Furthermore, PRK1 promotes a functional complex of AR with the co-activator TIF-2. Importantly, PRK signalling also stimulates AR activity in the presence of adrenal androgens, which are still present in prostate tumour patients subjected to testicular androgen ablation therapy. Moreover, PRK1 activates AR even in the presence of the AR antagonist cyproterone acetate that is used in the clinical management of prostate cancer. Since prostate tumours strongly overexpress PRK1, our data support a model in which AR activity is controlled by PRK signalling.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
Essential to salmonellae pathogenesis is an export device called the type III secretion system (TTSS), which mediates the transfer of bacterial effector proteins from the bacterial cell into the host cell cytoplasm. Once inside the host cell, these effectors are then capable of altering a variety of host cellular functions in order to promote bacterial survival and colonization. SspH1 is a Salmonella enterica serovar Typhimurium TTSS effector that localizes to the mammalian nucleus and down-modulates production of proinflammatory cytokines by inhibiting nuclear factor (NF)-kappaB-dependent gene expression. To identify mammalian binding partners of SspH1 a yeast two-hybrid screen against a human spleen cDNA library was performed. It yielded a serine/threonine protein kinase called protein kinase N 1 (PKN1). The leucine-rich repeat domain of SspH1 was demonstrated to mediate this interaction and also inhibition of NF-kappaB-dependent gene expression. This suggested that PKN1 may play a role in modulation of the NF-kappaB signalling pathway. Indeed, we found that expression of constitutively active PKN1 in mammalian cells results in a decrease, while depletion of PKN1 by RNA interference causes an increase in NF-kappaB-dependent reporter gene expression. These data indicate that SspH1 may inhibit the host's inflammatory response by interacting with PKN1.
Evidence
2:
Inferred from Physical InteractionIntAct
Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
Evidence
3:
Inferred from Physical InteractionIntAct
PKNalpha is a fatty acid- and Rho-activated serine/threonine protein kinase having a catalytic domain homologous to members of the protein kinase C family. Recently it was reported that PKNalpha is involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway. To date, however, how PKNalpha regulates the p38gamma MAPK signaling pathway is unclear. Here we demonstrate that PKNalpha efficiently phosphorylates MLTKalpha (MLK-like mitogen-activated protein triple kinase), which was recently identified as a MAPK kinase kinase (MAPKKK) for the p38 MAPK cascade. Phosphorylation of MLTKalpha by PKNalpha enhances its kinase activity in vitro. Expression of the kinase-negative mutant of PKNalpha inhibited the mobility shift of MLTKalpha caused by osmotic shock in SDS-PAGE. Furthermore, PKNalpha associates with each member of the p38gamma MAPK signaling pathway (p38gamma, MKK6, and MLTKalpha). These results suggest that PKNalpha functions as not only an upstream activator of MLTKalpha but also a putative scaffold protein for the p38gamma MAPK signaling pathway.
Evidence
4:
Inferred from Physical InteractionUniProtKB
Class IIa histone deacetylases (HDACs) -4, -5, -7 and -9 undergo signal-dependent nuclear export upon phosphorylation of conserved serine residues that are targets for 14-3-3 binding. Little is known of other mechanisms for regulating the subcellular distribution of class IIa HDACs. Using a biochemical purification strategy, we identified protein kinase C-related kinase-2 (PRK2) as an HDAC5-interacting protein. PRK2 and the related kinase, PRK1, phosphorylate HDAC5 at a threonine residue (Thr-292) positioned within the nuclear localization signal (NLS) of the protein. HDAC7 and HDAC9 contain analogous sites that are phosphorylated by PRK, while HDAC4 harbors a non-phosphorylatable alanine residue at this position. We provide evidence to suggest that the unique phospho-acceptor cooperates with the 14-3-3 target sites to impair HDAC nuclear import.
A novel protein kinase, designated PKN, was identified by molecular cloning from a human hippocampus cDNA library. PKN consists of 942 amino acids with a calculated molecular mass of 103,925 daltons. PKN has leucine zipper-like sequences in its amino terminal region and contains a catalytic domain that shows strong similarity to that of protein kinase C family. Northern blot analysis indicates PKN is expressed ubiquitously in human tissues. Antisera against PKN identified a 120K dalton protein on SDS polyacrylamide gel electrophoresis when PKN was expressed in the insect cells or COS7 cells. Recombinant PKN revealed an intrinsic protein kinase activity associated with a 120K protein. This activity was abolished by mutation of the lysine residue in the potential ATP binding site.
Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr(774) in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Czeta, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.
Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.
Class IIa histone deacetylases (HDACs) -4, -5, -7 and -9 undergo signal-dependent nuclear export upon phosphorylation of conserved serine residues that are targets for 14-3-3 binding. Little is known of other mechanisms for regulating the subcellular distribution of class IIa HDACs. Using a biochemical purification strategy, we identified protein kinase C-related kinase-2 (PRK2) as an HDAC5-interacting protein. PRK2 and the related kinase, PRK1, phosphorylate HDAC5 at a threonine residue (Thr-292) positioned within the nuclear localization signal (NLS) of the protein. HDAC7 and HDAC9 contain analogous sites that are phosphorylated by PRK, while HDAC4 harbors a non-phosphorylatable alanine residue at this position. We provide evidence to suggest that the unique phospho-acceptor cooperates with the 14-3-3 target sites to impair HDAC nuclear import.
Protein kinase C-related kinase 1 (PRK1 or PKN) is involved in regulation of the intermediate filaments of the actin cytoskeleton, as well as having effects on processes as diverse as mitotic timing and apoptosis. It is activated by interacting with the Rho family small G proteins and arachidonic acid or by caspase cleavage. We have previously shown that the HR1b of PRK1 binds exclusively to Rac1, whereas the HR1a domain binds to both Rac1 and RhoA. Here, we have determined the solution structure of the HR1b-Rac complex. We show that HR1b binds to the C-terminal end of the effector loop and switch 2 of Rac1. Comparison with the HR1a-RhoA structure shows that this part of the Rac1-HR1b interaction is homologous to one of the contact sites that HR1a makes with RhoA. The Rac1 used in this study included the C-terminal polybasic region, which is frequently omitted from structural studies, as well as the core G domain. The Rac1 C-terminal region reverses in direction to interact with residues in switch 2, and the polybasic region itself interacts with residues in HR1b. The interactions with HR1b do not prevent the polybasic region being available to contact the negatively charged membrane phospholipids, which is considered to be its primary role. This is the first structural demonstration that the C terminus of a G protein forms a novel recognition element for effector binding.
BACKGROUND: The Rho-related GTP-binding proteins Cdc42 and Rac1 have been shown to regulate signaling pathways involved in cytoskeletal reorganization and stress-responsive JNK (Jun N-terminal kinase) activation. However, to date, the GTPase targets that mediate these effects have not been identified. PAK defines a growing family of mammalian kinases that are related to yeast Ste20 and are activated in vitro through binding to Cdc42 and Rac1 (PAK: p21 Cdc42-/Rac-activated kinase). Clues to PAK function have come from studies of Ste20, which controls the activity of the yeast mating mitogen-activated protein (MAP) kinase cascade, in response to a heterotrimeric G protein and Cdc42. RESULTS: To initiate studies of mammalian Ste20-related kinases, we identified a novel human PAK isoform, hPAK1. When expressed in yeast, hPAK1 was able to replace Ste20 in the pheromone response pathway. Chemical mutagenesis of a plasmid encoding hPAK1, followed by transformation into yeast, led to the identification of a potent constitutively active hPAK1 with a substitution of a highly conserved amino-acid residue (L107F) in the Cdc42-binding domain. Expression of the hPAK1(L107F) allele in mammalian cells led to specific activation of the Jun N-terminal kinase MAP kinase pathway, but not the mechanistically related extracellular signal-regulated MAP kinase pathway. CONCLUSIONS: These results demonstrate that hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway in mammalian cells, as Ste20 does in yeast. Thus, PAK and Ste20 kinases play key parts in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation.
The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of detection of, or exposure to, a hyperosmotic environment, i.e. an environment with a higher concentration of solutes than the organism or cell.
Rho GTPases regulate multiple signal transduction pathways that influence many aspects of cell behaviour, including migration, morphology, polarity and cell cycle. Through their ability to control the assembly and organization of the actin and microtubule cytoskeletons, Rho and Cdc42 make several key contributions during the mitotic phase of the cell cycle, including spindle assembly, spindle positioning, cleavage furrow contraction and abscission. We now report that PRK2/PKN2, a Ser/Thr kinase and Rho/Rac effector protein, is an essential regulator of both entry into mitosis and exit from cytokinesis in HeLa S3 cells. PRK2 is required for abscission of the midbody at the end of the cell division cycle and for phosphorylation and activation of Cdc25B, the phosphatase required for activation of mitotic cyclin/Cdk1 complexes at the G2/M transition. This reveals an additional step in the mammalian cell cycle controlled by Rho GTPases.
The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.
In addition to the classical activation by ligands, nuclear receptor activity is also regulated by ligand-independent signalling. Here, we unravel a novel signal transduction pathway that links the RhoA effector protein kinase C-related kinase PRK1 to the transcriptional activation of the androgen receptor (AR). Stimulation of the PRK signalling cascade results in a ligand-dependent superactivation of AR. We show that AR and PRK1 interact both in vivo and in vitro. The transactivation unit 5 (TAU-5) located in the N-terminus of AR suffices for activation by PRK1. Thus, TAU-5 defines a novel, signal-inducible transactivation domain. Furthermore, PRK1 promotes a functional complex of AR with the co-activator TIF-2. Importantly, PRK signalling also stimulates AR activity in the presence of adrenal androgens, which are still present in prostate tumour patients subjected to testicular androgen ablation therapy. Moreover, PRK1 activates AR even in the presence of the AR antagonist cyproterone acetate that is used in the clinical management of prostate cancer. Since prostate tumours strongly overexpress PRK1, our data support a model in which AR activity is controlled by PRK signalling.
Evidence
2:
Inferred from Mutant PhenotypeUniProtKB
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
A novel protein kinase, designated PKN, was identified by molecular cloning from a human hippocampus cDNA library. PKN consists of 942 amino acids with a calculated molecular mass of 103,925 daltons. PKN has leucine zipper-like sequences in its amino terminal region and contains a catalytic domain that shows strong similarity to that of protein kinase C family. Northern blot analysis indicates PKN is expressed ubiquitously in human tissues. Antisera against PKN identified a 120K dalton protein on SDS polyacrylamide gel electrophoresis when PKN was expressed in the insect cells or COS7 cells. Recombinant PKN revealed an intrinsic protein kinase activity associated with a 120K protein. This activity was abolished by mutation of the lysine residue in the potential ATP binding site.
Posttranslational modifications of histones such as methylation, acetylation and phosphorylation regulate chromatin structure and gene expression. Here we show that protein-kinase-C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor target genes. PRK1 is pivotal to androgen receptor function because PRK1 knockdown or inhibition impedes androgen receptor-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation and inhibits androgen-induced demethylation of histone H3. Moreover, serine-5-phosphorylated RNA polymerase II is no longer observed at androgen receptor target promoters. Phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC)-domain-containing protein JMJD2C. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for gene activation, identifying PRK1 as a gatekeeper of androgen receptor-dependent transcription. Importantly, levels of PRK1 and phosphorylated H3T11 correlate with Gleason scores of prostate carcinomas. Finally, inhibition of PRK1 blocks proliferation of androgen receptor-induced tumour cell proliferation, making PRK1 a promising therapeutic target.
Kinase activity is activated upon binding to Rho proteins (RHOA, RHOB and RAC1). Activated by lipids, particularly cardiolipin and to a lesser extent by other acidic phospholipids. Activated by caspase-3 (CASP3) cleavage during apoptosis. Two specific sites, Thr-774 (activation loop of the kinase domain) and Ser-916 (turn motif), need to be phosphorylated for its full activation.
Proc. Natl. Acad. Sci. U.S.A. 95, 11566-11571 (1998)[PubMed:9751706]
PKN, a fatty acid- and Rho-activated serine/threonine kinase having a catalytic domain highly homologous to protein kinase C (PKC), was cleaved at specific sites in apoptotic Jurkat and U937 cells on Fas ligation and treatment with staurosporin or etoposide, respectively. The cleavage of PKN occurred with a time course similar to that of PKCdelta, a known caspase substrate. This proteolysis was inhibited by a caspase inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde. The cleavage fragments were generated in vitro from PKN by treatment with recombinant caspase-3. Site-directed mutagenesis of specific aspartate residues prevented the appearance of these fragments. These results indicate that PKN is cleaved by caspase-3 or related protease during apoptosis. The major proteolysis took place between the amino-terminal regulatory domain and the carboxyl-terminal catalytic domain, and it generated a constitutively active kinase fragment. The cleavage of PKN may contribute to signal transduction, eventually leading to apoptosis.
The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.
Protein involved in the transfer of genetic information from DNA to messenger RNA (mRNA) by DNA-directed RNA polymerase. In the case of some RNA viruses, protein involved in the transfer of genetic information from RNA to messenger RNA (mRNA) by RNA-directed RNA polymerase.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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