PKC-related serine/threonine-protein kinase and Rho/Rac effector protein that participates in specific signal transduction responses in the cell. Plays a role in the regulation of cell cycle progression, actin cytoskeleton assembly, cell migration, cell adhesion, tumor cell invasion and transcription activation signaling processes. Phosphorylates CTTN in hyaluronan-induced astrocytes and hence decreases CTTN ability to associate with filamentous actin. Phosphorylates HDAC5, therefore lead to impair HDAC5 import. Direct RhoA target required for the regulation of the maturation of primordial junctions into apical junction formation in bronchial epithelial cells. Required for G2/M phases of the cell cycle progression and abscission during cytokinesis in a ECT2-dependent manner. Stimulates FYN kinase activity that is required for establishment of skin cell-cell adhesion during keratinocytes differentiation. Regulates epithelial bladder cells speed and direction of movement during cell migration and tumor cell invasion. Inhibits Akt pro-survival-induced kinase activity. Mediates Rho protein-induced transcriptional activation via the c-fos serum response factor (SRF). Phosphorylates HCV NS5B leading to stimulation of HCV RNA replication.
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.
Rho GTPases regulate multiple signaling pathways to control a number of cellular processes during epithelial morphogenesis. To investigate the downstream pathways through which Rho regulates epithelial apical junction formation, we screened a small interfering RNA (siRNA) library targeting 28 known Rho target proteins in 16HBE human bronchial epithelial cells. This led to the identification of the serine-threonine kinase PRK2 (protein kinase C-related kinase 2, also called PKN2). Depletion of PRK2 does not block the initial formation of primordial junctions at nascent cell-cell contacts but does prevent their maturation into apical junctions. PRK2 is recruited to primordial junctions, and this localization depends on its C2-like domain. Rho binding is essential for PRK2 function and also facilitates PRK2 recruitment to junctions. Kinase-dead PRK2 acts as a dominant-negative mutant and prevents apical junction formation. We conclude that PRK2 is recruited to nascent cell-cell contacts through its C2-like and Rho-binding domains and promotes junctional maturation through a kinase-dependent pathway.
The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.
Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown.
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 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.
The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.
The Ras-related Rho family GTPases mediate signal transduction pathways that regulate a variety of cellular processes. Like Ras, the Rho proteins (which include Rho, Rac, and CDC42) interact directly with protein kinases, which are likely to serve as downstream effector targets of the activated GTPase. Activated RhoA has recently been reported to interact directly with several protein kinases, p120 PKN, p150 ROK alpha and -beta, p160 ROCK, and p164 Rho kinase. Here, we describe the purification of a novel Rho-associated kinase, p140, which appears to be the major Rho-associated kinase activity in most tissues. Peptide microsequencing revealed that p140 is probably identical to the previously reported PRK2 kinase, a close relative of PKN. However, unlike the previously described Rho-binding kinases, which are Rho specific, p140 associates with Rac as well as Rho. Moreover, the interaction of p140 with Rho in vitro is nucleotide independent, whereas the interaction with Rac is completely GTP dependent. The association of p140 with either GTPase promotes kinase activity substantially, and expression of a kinase-deficient form of p140 in microinjected fibroblasts disrupts actin stress fibers. These results indicate that p140 may be a shared kinase target of both Rho and Rac GTPases that mediates their effects on rearrangements of the actin cytoskeleton.
Akt is stimulated by several growth factors and has a major anti-apoptotic role in the cell. Therefore, we hypothesized that a pathway leading to the inhibition of Akt might be utilized in the process of apoptosis. Accordingly, we used a yeast two-hybrid screening assay to identify the proteins that interact with and possibly inhibit Akt. We found that the C-terminal region of protein kinase C-related kinase 2 (PRK2), containing amino acids 862 to 908, specifically binds to Akt in yeast and mammalian cells. During early stages of apoptosis, the C-terminal region of PRK2 is cleaved from the inhibitory N-terminal region and can bind Akt. The protein-protein interaction between Akt and the PRK2 C-terminal region specifically down-modulates the protein kinase activities of Akt by inhibiting phosphorylation at threonine 308 and serine 473 of Akt. This inhibition of Akt leads to the inhibition of the downstream signaling of Akt in vivo. The PRK2 C-terminal fragment strongly inhibits the Akt-mediated phosphorylation of BAD, a pro-apoptotic Bcl-2 family protein, and blocks the anti-apoptotic activities of Akt in vivo. These results provide direct evidence that the products of protein cleavage during apoptosis inhibit pro-survival signalings, leading to the amplification of pro-apoptotic signalings in the cell.
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.
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.
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
J. Biol. Chem. 274, 5542-5549 (1999)[PubMed:10026169]
Adapter proteins made up of Src homology (SH) domains mediate multiple cellular signaling events initiated by receptor protein tyrosine kinases. Here we report that Grb4 is an adapter protein closely related to but distinct from Nck that is made up of three SH3 domains and one SH2 domain. Northern analysis indicated that both genes are expressed in multiple tissues. Both Nck and Grb4 proteins could associate with receptor tyrosine kinases and the SH3-binding proteins PAK, Sos1, and PRK2, and they synergized with v-Abl and Sos to induce gene expression via the transcription factor Elk-1. Although neither protein was transforming on its own, both Nck and Grb4 cooperated with v-Abl to transform NIH 3T3 cells and influenced the morphology and anchorage-dependent growth of wild type Ras-transformed cells. Nck and Grb4 therefore appear to be functionally redundant.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.
Evidence
3:
Inferred from Physical InteractionUniProtKB
Rho GTPases regulate multiple signaling pathways to control a number of cellular processes during epithelial morphogenesis. To investigate the downstream pathways through which Rho regulates epithelial apical junction formation, we screened a small interfering RNA (siRNA) library targeting 28 known Rho target proteins in 16HBE human bronchial epithelial cells. This led to the identification of the serine-threonine kinase PRK2 (protein kinase C-related kinase 2, also called PKN2). Depletion of PRK2 does not block the initial formation of primordial junctions at nascent cell-cell contacts but does prevent their maturation into apical junctions. PRK2 is recruited to primordial junctions, and this localization depends on its C2-like domain. Rho binding is essential for PRK2 function and also facilitates PRK2 recruitment to junctions. Kinase-dead PRK2 acts as a dominant-negative mutant and prevents apical junction formation. We conclude that PRK2 is recruited to nascent cell-cell contacts through its C2-like and Rho-binding domains and promotes junctional maturation through a kinase-dependent pathway.
Eur. J. Biochem. 227, 344-351 (1995)[PubMed:7851406]
The cDNA clones for two members of a novel protein kinase family were isolated and sequenced. These protein-kinase-C-related kinases, PRK1 and PRK2, display extensive identity to each other, revealing non-kinase domain similar regions. HR1 and HR2. HR1 contains a motif repeated three times (HR1a-c), while HR2 shows similarity to the amino-terminal sequence of protein-kinase-C epsilon and eta isotypes. Both PRK1 and PRK2, expressed in COS 1 cells, are autophosphorylated in immunoprecipitates, indicating intrinsic kinase activity. PRK1 and PRK2, as well as a third member of this family, PRK3, show distinct patterns of expression in adult tissues.
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 formation of an apical junction, a functional unit located near the cell apex at the points of contact between epithelial cells composed of the tight junction, the zonula adherens junction and the desmosomes, by the aggregation, arrangement and bonding together of its constituents.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Rho GTPases regulate multiple signaling pathways to control a number of cellular processes during epithelial morphogenesis. To investigate the downstream pathways through which Rho regulates epithelial apical junction formation, we screened a small interfering RNA (siRNA) library targeting 28 known Rho target proteins in 16HBE human bronchial epithelial cells. This led to the identification of the serine-threonine kinase PRK2 (protein kinase C-related kinase 2, also called PKN2). Depletion of PRK2 does not block the initial formation of primordial junctions at nascent cell-cell contacts but does prevent their maturation into apical junctions. PRK2 is recruited to primordial junctions, and this localization depends on its C2-like domain. Rho binding is essential for PRK2 function and also facilitates PRK2 recruitment to junctions. Kinase-dead PRK2 acts as a dominant-negative mutant and prevents apical junction formation. We conclude that PRK2 is recruited to nascent cell-cell contacts through its C2-like and Rho-binding domains and promotes junctional maturation through a kinase-dependent pathway.
A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase, and an execution phase, which is triggered by the former.
The progression of biochemical and morphological phases and events that occur in a cell during successive cell replication or nuclear replication events. Canonically, the cell cycle comprises the replication and segregation of genetic material followed by the division of the cell, but in endocycles or syncytial cells nuclear replication or nuclear division may not be followed by cell division.
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.
Any process that activates or increases the frequency, rate or extent of the division of the cytoplasm of a cell, and its separation into two daughter cells, that occurs in the context of cell cycle progression.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
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.
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.
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.
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.
Kinase activity is activated upon binding to GTP-bound Rhoa/Rac1 GTPases. Activated by caspase-3 (CASP3) cleavage during apoptosis. Activated by lipids, particularly cardiolipin and to a lesser extent by other acidic phospholipids and unsaturated fatty acids. Two specific sites, Thr-816 (activation loop of the kinase domain) and Thr-958 (turn motif), need to be phosphorylated for its full activation.
PRK2/PKNgamma is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure-function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic competence. Moreover, the PRK2-Delta958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.
Akt is stimulated by several growth factors and has a major anti-apoptotic role in the cell. Therefore, we hypothesized that a pathway leading to the inhibition of Akt might be utilized in the process of apoptosis. Accordingly, we used a yeast two-hybrid screening assay to identify the proteins that interact with and possibly inhibit Akt. We found that the C-terminal region of protein kinase C-related kinase 2 (PRK2), containing amino acids 862 to 908, specifically binds to Akt in yeast and mammalian cells. During early stages of apoptosis, the C-terminal region of PRK2 is cleaved from the inhibitory N-terminal region and can bind Akt. The protein-protein interaction between Akt and the PRK2 C-terminal region specifically down-modulates the protein kinase activities of Akt by inhibiting phosphorylation at threonine 308 and serine 473 of Akt. This inhibition of Akt leads to the inhibition of the downstream signaling of Akt in vivo. The PRK2 C-terminal fragment strongly inhibits the Akt-mediated phosphorylation of BAD, a pro-apoptotic Bcl-2 family protein, and blocks the anti-apoptotic activities of Akt in vivo. These results provide direct evidence that the products of protein cleavage during apoptosis inhibit pro-survival signalings, leading to the amplification of pro-apoptotic signalings in the cell.
J. Biol. Chem. 272, 29449-29453 (1997)[PubMed:9368003]
The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translated in vitro in the presence of [35S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp117 and Asp700 in vitro. In addition, PRK2 is cleaved rapidly during Fas- and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein involved in the complex series of events by which the cell duplicates its contents and divides into two. The eukaryotic cell cycle can be divided in four phases termed G1 (first gap period), S (synthesis, phase during which the DNA is replicated), G2 (second gap period) and M (mitosis). The prokaryotic cell cycle typically involves a period of growth followed by DNA replication, partition of chromosomes, formation of septum and division into two similar or identical daughter cells.
Protein involved in the separation of one cell into two daughter cells. In eukaryotic cells, cell division includes the nuclear division (mitosis) and the subsequent cytoplasmic division (cytokinesis).
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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