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PRAK, a novel protein kinase regulated by the p38 MAP kinase

1998; Springer Nature; Volume: 17; Issue: 12 Linguagem: Inglês

10.1093/emboj/17.12.3372

1460-2075

Liguo New,

Cellular Mechanics and Interactions

Article15 June 1998free access PRAK, a novel protein kinase regulated by the p38 MAP kinase Liguo New Liguo New Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Yong Jiang Yong Jiang Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Ming Zhao Ming Zhao Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Kang Liu Kang Liu Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Wei Zhu Wei Zhu Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Laura J. Flood Laura J. Flood Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Yutaka Kato Yutaka Kato Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Graham C.N. Parry Graham C.N. Parry Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Jiahuai Han Corresponding Author Jiahuai Han Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Liguo New Liguo New Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Yong Jiang Yong Jiang Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Ming Zhao Ming Zhao Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Kang Liu Kang Liu Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Wei Zhu Wei Zhu Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Laura J. Flood Laura J. Flood Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Yutaka Kato Yutaka Kato Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Graham C.N. Parry Graham C.N. Parry Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Jiahuai Han Corresponding Author Jiahuai Han Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Author Information Liguo New1, Yong Jiang1, Ming Zhao1, Kang Liu1, Wei Zhu1, Laura J. Flood1, Yutaka Kato1, Graham C.N. Parry1 and Jiahuai Han 1 1Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037 USA ‡L.New and Y.Jiang contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3372-3384https://doi.org/10.1093/emboj/17.12.3372 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20–30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38α and p38β both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo. Introduction A variety of extracellular stimuli produce cellular responses via activation of the mitogen-activated protein (MAP) kinase cascades (Davis, 1993; Waskiewicz and Cooper, 1995; Su and Karin, 1996; Fanger et al., 1997; Robinson and Cobb, 1997). The specificity of the cellular response is determined by the activation of a particular MAP kinase pathway in response to a given stimulus and by the activation of downstream targets by a given MAP kinase. MAP kinase family members have been found to regulate diverse biological functions by phosphorylation of specific target molecules found within the cell membrane, cytoplasm and nucleus, and thereby participate in the regulation of a variety of cellular processes including cell proliferation, differentiation and immune responses (Blenis, 1993; Marshall, 1994; Cano and Mahadevan, 1995; Seger and Krebs, 1995). p38 (or p38α, also known as CSBP and RK) is a MAP kinase superfamily member which was first identified and cloned as an intracellular protein rapidly tyrosine phosphorylated upon treatment of macrophages with lipopolysaccharides (Han et al., 1993, 1994). p38α was also cloned as a specific target of pyridinyl imidazole derivatives such as SB203580 which inhibit the production of proinflammatory cytokines by monocytes (Lee et al., 1994). Three closely related kinases of p38 subsequently were cloned and characterized: p38β (Jiang et al., 1996), p38γ (also known as ERK6 or SAPK3) (Lechner et al., 1996; Li et al., 1996; Mertens et al., 1996) and p38δ (also known as SAPK4) (Goedert et al., 1997; Jiang et al., 1997; Wang et al., 1997). p38α and p38β are sensitive to SB203580 inhibition, but the activities of p38γ and p38δ are unaffected (Goedert et al., 1997). The entire family of p38 MAP kinases can be activated by osmotic changes in the extracellular environment. Other stimuli, such as UV light, oxidation and proinflammatory cytokines, are potent activators of specific p38 family members (Raingeaud et al., 1995; Jiang et al., 1997). In addition, some growth factors, such as erythropoietin and interleukin-3 (IL-3), are activators of p38α in certain cell types (Foltz et al., 1997; Nagata et al., 1997). All four p38 family members are activated by MAP kinase kinase (MKK) 3 or MKK6 via phosphorylation of the TGY motif (Derijard et al., 1995). Several proteins have been identified as substrates of p38α including transcription factors (Price et al., 1996; Wang and Ron, 1996; Han et al., 1997; Janknecht and Hunter, 1997; Whitmarsh et al., 1997) and protein kinases (Rouse et al., 1994; Ludwig et al., 1996; McLaughlin et al., 1996; Fukunaga and Hunter, 1997; Waskiewicz et al., 1997). Protein kinase substrates of MAP kinases are believed to play an important role in amplifying and diversifying MAP kinase signals. Three groups of kinases have been shown to be activated by MAP kinases, ribosomal S6 kinase (RSK also known as MAPKAP-K1), MAP kinase-activated protein kinase (MAPKAP-K) and MAP kinase-interacting kinase or MAP kinase signal-integrating kinase (MNK) (Rouse et al., 1994; Zhao et al., 1996; Fukunaga and Hunter, 1997; Waskiewicz et al., 1997). The activity of RSK proteins is regulated by the ERK kinase pathway, and these proteins play important roles in a variety of processes including cell proliferation (Jones et al., 1988; Blenis, 1993; Zhao et al., 1996). The two known MAPKAP-K family members, MAPKAP-K2 and MAPKAP-K3, can be activated by p38α in vitro and their activation can be inhibited in vivo by SB203580, a specific inhibitor of p38α and p38β (McLaughlin et al., 1996). In turn, activated MAPKAP-K2/3 can phosphorylate small heat shock protein 27 (HSP27) (Stokoe et al., 1992b), lymphocyte-specific protein 1 (LSP1) (Huang et al., 1997a), cAMP response element-binding protein (CREB), ATF1 (Tan et al., 1996) and tyrosine hydroxylase (Thomas et al., 1997). A third group of MAP kinase-regulated protein kinases, MNK, was identified recently (Fukunaga and Hunter, 1997; Waskiewicz et al., 1997). These kinases are regulated by p38 and ERK, but not by JNK. MNK1/2 can phosphorylate eukaryotic initiation factor-4E (eIF-4E) in vitro, which suggests an important link between MAP kinase activation and translational initiation (Waskiewicz et al., 1997). Despite the identification of several protein kinases as substrates for p38, there is evidence of additional downstream protein kinases. For example, Iordanov et al. reported that short wavelength UV irradiation (UVC) elicits p38-dependent CREB phosphorylation, and that the phosphorylation was mediated by an unknown p38-activated protein kinase (Iordanov et al., 1997). To understand better the regulation and function of the p38 pathways, it is necessary to identify and characterize substrates of p38. Here, we describe the structure, function and regulation of a new protein kinase, p38-regulated/activated kinase (PRAK). This enzyme is activated by stress-related extracellular stimuli. The regulatory phosphorylation site of PRAK is Thr182 and this residue is phosphorylated by p38α and p38β in vitro. Activated PRAK specifically phosphorylates HSP27 at its physiologically relevant sites in vitro. In-gel kinase assays indicated that PRAK is one of the major stress-activated HSP27 kinases in whole-cell extracts from HeLa cells, suggesting that PRAK has a potential role in mediating stress-induced small heat shock protein phosphorylation in vivo. Results Molecular cloning of human PRAK Although the RSK and MAPKAP-K share only 35% amino acid sequence identity, a common feature of these MAP kinase-regulated kinases was identified. Each contains the phosphorylation site LX*TP located within the T-loop (also called Loop 12) between kinase domains VII and VIII. The sequences adjacent to this phosphorylation site are also conserved within the RSK and MAPKAP-K proteins. We hypothesized that there may be additional MAP kinase-regulated protein kinases that contain conserved LXTP sites and distinct flanking sequences. The peptide sequences LXTPCYTPYYVAP and LXTPCYTANFVAP from MAPKAP-K and RSK respectively were used to search the dbest database using the tblastn program. Seven expressed sequence tag (EST) clones were identified that contained the LXTP site and novel flanking sequences. The sequence LTTPCGSAEYMAP was present in four EST clones (DDBJ/EMBL/GenBank accession Nos AA080236, W24753, AA083972 and AA157440). The sequence LXTPQFTPYYVAP was found in a single EST clone (DDBJ/EMBL/GenBank accession No. R68329) and the sequence LKTPCFTLHYAAP was present in two clones (DDBJ/EMBL/GenBank accession Nos N57096 and H09985). Additional sequences of these EST clones indicated that they are cDNAs for novel protein kinases. We hypothesized that these clones represent three new protein kinase groups that are regulated by MAP kinases. The EST clones of DDBJ/EMBL/GenBank accession Nos AA157440, R68329 and N57096 were sequenced completely and used to screen a human placental cDNA library to isolate full-length cDNA clones. While this study was in progress, two new protein kinases were reported by Waskiewicz et al. (1997) and Fukunaga and Hunter (1997), termed MNK1 and 2, that contained the sequence LXTPCGSAEYMAP. These protein kinases appear to belong to a new protein kinase group because they have only 30% identity to the RSK and MAPKAP-K. Importantly, this group of protein kinases is regulated by MAP kinases as our hypothesis would predict (Fukunaga and Hunter, 1997; Waskiewicz et al., 1997). Because the available evidence suggests our approach may identify new kinase substrates of MAP kinases, we focused our efforts on the cDNA clones isolated from the human placental cDNA library that encode a LXTPQFTPYYVAP sequence. Complete sequencing of the three isolated clones revealed that they all encoded a single protein. The nucleotide sequence of the longest clone is shown in Figure 1. No in-frame stop codon was found at the 5′ end of the clone. 5′-RACE was performed but no additional 5′ sequence was obtained. Therefore, we tentatively assigned the first ATG as the start codon. The cDNA has an open reading frame encoding a protein of 471 amino acids with a calculated molecular mass of 54 kDa (Figure 1). This is consistent with the molecular mass of the endogenous protein on Western blots (Figure 3, see below). Since this protein kinase is regulated/activated specifically by p38 (see below), we named this kinase p38-regulated/activated kinase (PRAK). PRAK contains the conserved protein kinase domains I–XI which are characteristic of all protein kinases (Figure 1). A DDBJ/EMBL/GenBank database search revealed that PRAK is most closely related to the MAPKAP-K, MNK and RSK kinases. A sequence alignment of PRAK with a member of the RSK group, MAPKAP-K group and MNK group proteins is shown in Figure 2A. PRAK is 32.8, 33.1, 22.8, 25.4, 22.7, 20.0 and 20.1% identical to MAPKAP-K2, MAPKAP-K3, MNK1, MNK2, ISPK1 (RSK1), RSK2 and RSK3, respectively. A computer-generated diagram showing the relatedness of these protein kinases is shown in Figure 2B. The relatively low homology of this protein to the known MAP kinase-regulated protein kinases supports our prediction that the protein containing LXTPQFTPYYVAP may belong to a new kinase group. The tissue distribution of PRAK mRNA was assessed by Northern blot hybridization (Figure 3A). The PRAK transcript is ∼2.4 kb and is present in all eight tissues examined (Figure 3A). PRAK protein expression was analyzed in five different human cell lines by Western blot using an anti-PRAK antibody. The protein was detected in all of the cell lines as a 54 kDa protein, consistent with the predicted molecular weight (Figure 3B). Figure 1.The deduced amino acid and nucleotide sequence of human PRAK. The 11 protein kinase subdomains are indicated under the protein sequence with Roman numerals. The potential phosphorylation site (Thr182) of PRAK is marked by a solid triangle. The asterisk indicates a stop codon. The cDNA sequence of PRAK has been deposited in the DDBJ/EMBL/GenBank database under the accession number AF032437. Download figure Download PowerPoint Figure 2.(A) Sequence comparison of human PRAK, MAPKAP-K2, MNK1 and the C-terminal kinase domain of RSK2 using the Lasergene Megaline program (DNAStar, Madison, WI). Residues conserved between two or more protein kinases are boxed. (B) The ancestral relationships among human PRAK, human MAPKAP-K2, human MAPKAP-K3, human MNK1, murine MNK2, human RSK1(ISPK1), human RSK2, and human RSK3 are presented as a phylogenetic tree created by the Lasergene Megaline program. The scale beneath the tree measures the distance between sequences, and units indicate substitution events. Download figure Download PowerPoint Figure 3.Expression of PRAK. (A) A blot containing 2 μg of poly(A)+ RNA isolated from various human tissues was hybridized with a probe specific to PRAK. (B) Equal amounts of cell-free lysates from 293, A549, HeLa, HepG2 and Jurkat cells were separated by 12% SDS–PAGE and transferred onto a nitrocellulose membrane. PRAK was detected using a specific polyclonal antibody. Download figure Download PowerPoint HSP27 is a preferred substrate for PRAK in vitro To identify the substrate of PRAK, we tested a panel of proteins which are known substrates of PRAK-related protein kinases. As shown in Figure 4B, HSP27 and HSP25 are good substrates of PRAK in vitro. Phosphorylation of HSP27 and HSP25 by PRAK is similar in magnitude to that seen with MAPKAP-K2 when equal amounts of fully activated kinase were used in in vitro kinase assays (compare Figure 4B and C). Both PRAK and MAPKAP-K2 can phosphorylate glycogen synthetase, and myosin heavy chain to a lesser extent. PHAS-1, MBP, eIF-4E and the N-terminal portions of c-Jun (1–93) and ATF2 (1–109) were poor substrates for PRAK and MAPKAP-K2. To determine if PRAK is a major kinase for HSP27 in stress-activated cells, an in-gel kinase assay using recombinant HSP27 as substrate was performed (Figure 5, right panel). There are four major kinases in HeLa cell lysates that phosphorylate HSP27. Stimulation of HeLa cells with arsenite resulted in activation of the 45 and 54 kDa HSP27 kinases. Depletion of PRAK in cell lysates with an anti-PRAK polyclonal antibody specifically removed the kinase activity associated with the 54 kDa protein, while the other HSP27 kinases were unaffected. These data strongly suggested that PRAK was a major stress-activated kinase that can phosphorylate HSP27. Western blot analysis (Figure 5, left panel), revealed that the stress-activated 45 kDa HSP27 kinase in HeLa cell lysates is MAPKAP-K2. MAPKAP-K2 has been reported to phosphorylate serines 15, 78 and 82 of HSP27 in response to stress or mitogens in vivo (Stokoe et al., 1992b; Knauf et al., 1994). Therefore, we performed an experiment to determine whether or not PRAK phosphorylates HSP27 at the same sites as MAPKAP-K2. Recombinant HSP27 treated with PRAK or MAPKAP-K2 in the presence of [γ-32P]ATP was used for tryptic phosphopeptide mapping. As shown in Figure 6, PRAK and MAPKAP-K2 phosphorylated the same peptides. Thus PRAK, like MAPKAP-K2, phosphorylates HSP27 at the functionally relevant sites. The overlapping specificity of the two kinases, MAPKAP-K2 and PRAK, is not without precedent (Whitmarsh et al., 1995; Price et al., 1996; Janknecht and Hunter, 1997). However, the physiological relevance of this overlap requires further investigation. Figure 4.HSP25 and HSP27 are preferred substrates for PRAK in vitro. (A) The Coomassie Blue stain of the protein substrates used in the kinase assays. Approximately 10 μg of GST–c-Jun (1–93), GST–ATF2 (1–109), PHAS-1, HSP25, HSP27, MBP, eIF-4E, glycogen synthetase and myosin heavy chain were used as substrates, and 0.5 μg of fully activated PRAK (B) or MAPKAP-K2 (C) were used as kinases for in vitro kinase assays. The kinase reactions were stopped by adding SDS sample buffer, and reaction products were analyzed by SDS–PAGE. Substrate phosphorylation was detected by autoradiography, and was quantified by phosphoimaging. Comparable results were obtained in two independent experiments. Download figure Download PowerPoint Figure 5.PRAK is a HSP27 kinase activated by arsenite treatment in HeLa cells. In-gel kinase assay using HSP27 polymerized inside SDS–PAGE. The cell lysates of HeLa cells treated with or without 200 μM arsenite for 30 min were analyzed. Depletion of PRAK in the cell lysate was achieved by using anti-PRAK polyclonal antibody. The positions of PRAK and MAPKAP-K2 were determined by Western blotting as shown in the right hand panel. Download figure Download PowerPoint Figure 6.Tryptic phosphopeptide map of HSP27 phosphorylated by activated GST–PRAK and GST–MAPKAP-K2 in vitro. The panel marked as MIX indicates a mixture of equal amounts of HSP27 proteins phosphorylated by GST–PRAK and GST–MAPKAP-K2 prior to tryptic phosphopeptide mapping. The + sign on each panel indicates the original sample loading site. The perpendicular arrowheads indicate two-dimensional separations of phosphopeptides by electrophoresis and chromatography. Download figure Download PowerPoint PRAK is activated by environmental stress and proinflammatory cytokines A variety of agonists were tested for their ability to activate endogenous PRAK in HeLa cells. PRAK activity was measured using an immunokinase assay with HSP27 as the substrate. A Western blot demonstrated that equal amounts of PRAK were used in each assay (data not shown). As shown in Figure 7A, PRAK was strongly activated by stress stimuli such as arsenite, anisomycin and H2O2, and by the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Phorbol-12-myristate-13acetate (PMA) and calcium ionophore A23187 also activated PRAK but to a lesser extent. In contrast, IL-6 had no effect on PRAK activation in HeLa cells. Growth-related stimuli epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and serum had no or only modest effects on PRAK activity in HeLa cells (Figure 7B). A similar activation profile was obtained when transiently expressed hemagglutinin (HA)-tagged PRAK was assayed by immunokinase assay using anti-HA monoclonal antibody 12CA5 (data not shown). Figure 7.Activation of endogenous PRAK by various stimuli. HeLa cells were cultured in DMEM with 10% FBS. Approximately 106 cells (per sample) were treated with the Ca2+ ionophore A23187 (5 μM), PMA (100 nM), EGF (1 ng/ml), IL-6 (250 U/ml), TNF-α (100 ng/ml), H2O2 (400 μM), anisomycin (50 ng/ml), arsenite (200 μM) (A), or EGF (1 ng/ml), bFGF (10 ng/ml), PDGF-BB (10 ng/ml) and 20% serum (B) for 20 min at 37°C. Cells were serum starved for 5 h before the stimulation with growth factors. The kinase activity of PRAK isolated by immunoprecipitation with anti-PRAK antiserum was measured using HSP27 as the substrate. The fold activation by the different stimuli was calculated by dividing the radioactive intensity of phosphorylated HSP27 for each treatment by the untreated control and is shown under each lane. Similar results were obtained in two experiments. Download figure Download PowerPoint PRAK is a physiological substrate for p38α and p38β PRAK was cloned because it contained a putative phosphorylation site for MAP kinases. The data presented above demonstrate that PRAK is activated by a panel of stimuli that are known to activate p38 and c-Jun N-terminal kinase [JNK also known as stress-activated protein kinase (SAPK)]. We therefore hypothesized that PRAK is a downstream target of the p38 or JNK signaling pathways. To test this hypothesis, we performed an in vitro kinase assay using recombinant PRAK as the substrate and extracellular signal-regulated kinase 2 (ERK2), JNK2, p38α, p38β, p38γ or p38δ as the kinase (Figure 8). Equal amounts of each recombinant MAP kinase, activated by the appropriate upstream kinase (see Materials and methods), were used (Figure 8A). p38α, p38β, p38γ and p38δ showed higher activity toward PRAK in comparison with JNK2 and ERK2 (Figure 8B). Coupled kinase assays were performed to address whether the phosphorylation of PRAK by these MAP kinases influenced intrinsic PRAK activity using HSP27 as the substrate. PRAK activity was dramatically increased when it was phosphorylated by p38α or p38β (Figure 8C). In contrast, the enhancement of PRAK activity by p38γ, p38δ, JNK2 or ERK2 was not significant. Interestingly, p38δ efficiently phosphorylated PRAK (Figure 8B), but did not lead to activation of PRAK activity (Figure 8C). The inability of p38δ to activate PRAK is probably due to its failure to phosphorylate the regulatory site(s) of PRAK (see below). Figure 8.Phosphorylation and activation of PRAK by different MAP kinases in vitro. (A) Coomassie Blue stain of recombinant MAP kinases used in the experiments. (B) Phosphorylation of PRAK (5 μg) by different MAP kinases (0.5 μg) that were activated in vitro by MKKs as described in Materials and methods. The phosphorylated products were resolved by SDS–PAGE and quantified by phosphoimaging. The relative phosphorylation of PRAK by each MAP kinase was calculated by subtracting the intensity of PRAK autophosphorylation from that of each MAP kinase-treated PRAK. The result is shown as a bar graph. (C) Activation of PRAK by different MAP kinases was assessed by in vitro coupled kinase assays. Several paired kinase reactions, containing equal amounts of each MAP kinase (0.5 μg) and 10 μg of HSP27 in the presence or absence of 2 μg of GST–PRAK, were performed. The phosphorylated products were resolved by SDS–PAGE and quantified by phosphoimaging. The fold activation of PRAK by p38α, p38β, p38γ, p38δ, JNK2 and ERK2 was determined by the formula: [(radioactive intensity of HSP27 phosphorylated by a given MAP kinase activated PRAK and this MAP kinase) − (radioactive intensity of HSP27 phosphorylated by the given MAP kinase alone)]/(radioactive intensity of HSP27 phosphorylated by untreated PRAK). Comparable results were obtained in two independent experiments. Download figure Download PowerPoint Next, we determined if PRAK was downstream of the p38 pathway in intact cells. Substitution of the phosphorylation sites of MKK with glutamic acid (E) or aspartic acid (D) results in constitutively active forms of MKKs. Previously, we reported that expression of constitutively active MKK by recombinant adenovirus could activate a given MAP kinase pathway in intact cells (Huang et al., 1997b). Here, the same strategy was used independently to activate the p38, JNK or ERK pathway by expression of MKK1(E), MKK6(E) or MKK7(D). Twenty four hours after cells were infected with recombinant viruses, endogenous PRAK activity was measured using an immunokinase assay (Figure 9). Expression of MKK6(E), which activates the p38 signaling pathway, caused endogenous p38α/β and PRAK activation, supporting the concept that PRAK is regulated by the p38 pathway. In contrast, MKK7(D) expression activated JNK1 but not PRAK, and expression of MKK1(E) enhanced ERK2 activity but had no influence on PRAK activity. Therefore, JNK1 and ERK2 are unable to activate PRAK in vivo. Since MKK1 and MKK7 activate all ERK and JNK isoforms (Cowley et al., 1994; Marshall, 1994; Holland et al., 1997; Lu et al., 1997; Tournier et al., 1997), these data suggest that ERK1 and JNK2/3 are also unable to activate PRAK. Thus, PRAK is regulated specifically by the p38 pathway in intact cells. Figure 9.Effect of individual MAP kinase pathways on PRAK activity in intact cells. The constitutively active form of MAP kinase kinase, MKK1(E), MKK6(E) or MKK7(D) was expressed in HeLa cells (∼106 per sample) using an adenovirus-mediated gene delivery system. The control infection was done with an adenovirus encoding β-galactosidase. An m.o.i. of 10 was used for each of the recombinant viruses, because nearly 100% infection efficiency was achieved at this titer. After 24 h, the cells were lysed and PRAK was immunoprecipitated using an anti-PRAK antibody. The lysates subsequently were used to immunoprecipitate p38α/β, ERK2, p38α/β or JNK1 (corresponding to the lanes in the figure) from the cells expressing β-galactosidase, MKK1(E), MKK6(E) or MKK7(D), respectively. The upper panel shows endogenous PRAK activity from different cell lysates measured by immunokinase assays using HSP27 as the substrate. The middle panel shows that equal amounts of PRAK determined by Western blotting were used for the immunoprecipitation. The lower panel shows endogenous activities of p38α/β, ERK2, p38α/β or JNK2 (corresponding to the lanes in figure) of the cells infected by the control virus, ad-MKK1(E), ad-MM6(E) or ad-MKK7(D). GST–ELK1 was used as the ERK substrate, GST–ATF2 was used as the substrate for p38 and JNK. Comparable results were obtained in duplicate experiments. Download figure Download PowerPoint To test further whether PRAK is regulated by p38α/β under physiological conditions, arsenite, TNF-α and PMA were used to stimulate HeLa cells that had been pre-treated with a specific inhibitor for p38α/β, SB203580 (Figure 10). Blocking of p38α/β signaling dramatically inhibited arsenite- and TNF-α-, as well as PMA-induced PRAK activation. In contrast, treatment of cells with PD98059 at a concentration (10 μM) that inhibits PMA-induced ERK activation by 50% in HeLa cells (data not shown) had no effect on stress-induced PRAK activation, indicating that the ERK pathways does not play a role in regulating PRAK activity in HeLa cells. Taken together, these data indicate that p38α and p38β are in vivo regulators of PRAK. Figure 10.Effects of SB203580 and PD98059 on endogenous PRAK activity in the cells treated with TNF-α, arsenite or PMA. HeLa cells (∼106 per sample) were pre-treated with PD98059 (10 μM) or SB203580 (10 μM) for 30 min, and then stimulated with TNF-α (100 ng/ml), arsenite (200 μM) or PMA (100 nM) for 20 min. PRAK activities from these different treatments were measured by immunokinase assays with HSP27 as the substrate. The + or − under each lane indicates the presence or absence of a specific treatment. Comparable results were obtained in three experiments. Download figure Download PowerPoint To evaluate further the role of p38α and p38β in stress-induced PRAK activation, a series of experiments was performed to determine if p38 activation correlated with PRAK activation. HeLa cells were stimulated with increasing doses of TNF-α for 20 min or treated with 50 ng/ml of TNF-α for different periods of time. The activities of PRAK and p38α/β were measured by immunokinase assays. As shown in Figure 11A and B, there is a good correlation between TNF-α-induced activation of both PRAK and p38. Moreover, TNF-induced PRAK activation was inhibited in a dose-dependent manner by the p38α/β inhibito