Mitogen- and Stress-activated Protein Kinase 1 Mediates Activation of Akt by Ultraviolet B Irradiation*

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways.

teins Ras and Rac (9,10). The serine-threonine kinase Akt (also known as protein kinase B (PKB) 1 ) is activated by a wide variety of growth stimuli, including epidermal growth factor and insulin through phosphatidylinositol 3-kinase (PI3-K) (11,12). Ras activates the PI3-K/Akt signaling pathway by interacting directly with the p110 catalytic subunit of PI3-K (13). In addition, Kabuyama et al. (14) reported that UVB irradiation activates PI3-K in human fibroblast cells. These findings suggest that UV irradiation induces the activation of Akt. In a wide range of cellular systems, Akt has been shown to control intracellular pathways responsible for preventing cell death in response to a variety of extracellular stimuli (15)(16)(17). Furthermore, recent work has shown that Akt is not only a "cell survival" kinase but it may play an important role in protein synthesis, glycogenesis, and regulation of cell cycle progression (18,19). In contrast, identification of the gene encoding Akt as a transforming oncogene that causes thymic lymphomas in mice suggests a role for Akt in tumorigenesis (20). Overexpression of Akt has been demonstrated in a large proportion of ovarian (21), pancreatic (22), and breast cancers (23) in humans. Mutations of tumor suppressor gene Phosphatase and Tensin (PTEN), which directly antagonizes PI3-K, have been observed in a number of human cancers (24,25). The alteration of PTEN causes elevated phosphorylation of Akt (26 -28). A part of the tumor-promoting effects of UV irradiation may occur through activation of Akt. UVB plays a major role in development of human skin cancer (29,30), and UV irradiation has been shown to act both as a tumor initiator and a tumor promoter in animals (31,32). However, whether UV irradiation activates Akt is unclear.
In quiescent or serum-starved cells, Akt resides within the cytosol in a catalytically inactive state. Following stimulation of cells extracellularly with (e.g.) growth factors or cytokines, Akt is recruited to the plasma membrane and catalytically activated by phosphorylation at threonine 308 and serine 473 (19,(33)(34)(35). Phosphorylation of Akt at threonine 308 is catalyzed by the ubiquitously expressed and constitutively activated phosphatidylinositol 3,4,5-triphosphate-dependent protein kinase-1 (PDK-1) (34,35). The kinase responsible for phosphorylation of Akt at serine 473 has not been established definitively, although possible candidates have been proposed (36 -38).
Mitogen-activated protein (MAP) kinases belong to a large family of serine/threonine protein kinases and include extracellular signal-regulated protein kinases (Erks), p38 kinase, and c-Jun N-terminal kinases (JNKs) (39 -41). Generally, JNKs and p38 kinase are known to be activated by various forms of stress, such as UV irradiation, heat shock, and inflammation (40 -43). Our studies and those of others have shown that Erks are critical for UV-induced signal transduction (44 -46). Rane et al. (38) recently showed that the p38 kinase pathway regulates Akt activation in human neutrophils. The Erk signaling pathway also may be associated with anti-apoptotic effects (47,48). Therefore, we investigated the involvement of the MAP kinase signaling pathways in UVB-induced Akt acti-vation. In this study, we demonstrated that activation of Akt is induced by UVB irradiation and that the activation of Akt is mediated by Erks and p38 kinase through their downstream kinase, mitogen-and stress-activated protein kinase 1 (Msk1), in addition to the PI3-K/PDK pathway.

EXPERIMENTAL PROCEDURES
Materials-Fetal bovine serum (FBS) was from Gemini Bio-Product (Calabasas, CA); gentamicin was from BioWhittaker, Inc. (Walkersville, MD); Eagle's minimal essential medium (MEM) and L-glutamine were from Life Technologies, Inc.; the MAP kinase/Erk kinase 1 specific inhibitor PD 98059 and the PI3-K inhibitor LY 29402 were from BI-OMOL Research Laboratories, Inc. (Plymouth Meeting, PA); the p38 kinase inhibitor SB 202190 was from Calbiochem (La Jolla, CA); Akt fusion protein, radioactive and nonradioactive Akt immunoprecipitation kinase assay kit, anti-Msk1 antibody, anti-MAP kinase-activated FIG. 1. Induction of Akt phosphorylation by UVB irradiation. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were either exposed to UVB (4 kJ/m 2 ) or not and were subsequently cultured for the indicated time periods. The cells were lysed, and the phosphorylation levels of Akt at Thr-308 or Ser-473 were assessed as described under "Experimental Procedures." The cells were irradiated with UVB (4 kJ/m 2 ) and subsequently cultured for 30 min. The cells were lysed, and the phosphorylation levels of Akt were assessed by immunoblotting. B, lysates were prepared from the cells treated as described above, and Akt was immunoprecipitated using anti-Akt1/ PKB␣, PH domain antibody. The activity of Akt was measured using Akt substrate peptide and [␥-32 P]ATP. Columns and bars indicate the mean Ϯ S.E. of at least three experiments. C, lysates were prepared from the cells treated as described above, and Akt was immunoprecipitated using anti-Akt1/PKB␣, PH domain antibody. The activity of Akt was assessed using Bad fusion protein as a substrate, and the phosphorylation of Bad at Ser-136 was assessed by immunoblotting.
UV Irradiation-UVB irradiation was performed on serum-starved monolayer cultures utilizing a transluminator emitting UVB (54). The source of UVB was a bank of four Westinghouse F520 Lamps (National Biological, Twinsburg, OH) at 6 J/S/m light in the UVB range. Approximately 10% of the remaining radiation from the F520 lamp is in the UVA region (320 nm). Although almost no UVC leakage occurred, the UVB irradiation was carried out in a UVB exposure chamber fitted with a Kodak Kodacel K6808 filter that eliminates all wavelengths below 290 nm. This lamp is one of the most frequently used UVB sources for the study of carcinogenesis. The International Agency for Research on Cancer refers to this lamp as a source emitting mainly UVB irradiation for the study of cancer induction in animals. UVB irradiation was measured using the UVX radiometer from UVP (UVX-31).
Cell Generation of Stable Cotransfectants-The dominant negative mutants of Erk2, p38 kinase, or JNK1 were subcloned into a mammalian expression vector plasmid, CMV-neo. A dominant negative mutant plasmid of PI3-K p85 subunit or Msk1 C-terminal kinase-dead mutant plasmid or its control vector CMV5 plasmid was transfected or cotransfected with CMV-neo in JB6 Cl 41 cells by using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructors. The stable transfectants were obtained by selection for G418 resistance (300 g/ml) and further confirmed by assay of respective activities (44,(55)(56)(57).
Immunoblotting-Immunoblotting was carried out as described previously (44,55,56). In brief, JB6 Cl 41 cells and their stable transfectants were cultured to 80% confluence. The cells were starved in 0.1% FBS MEM for 48 h at 37°C. Then, medium was changed to fresh 0.1% FBS MEM, and cells were incubated for another 2-4 h at 37°C. Before the cells were treated with UVB or insulin, they were treated with or without LY 294002, PD 98059, or SB 202190 for 1 h. Then, the cells were exposed to UVB (4 kJ/m 2 ) and subsequently incubated for the indicated periods or for 30 min or treated with insulin (2.5 g/ml) for the indicated periods or for 5 min at 37°C in the presence of inhibitors. The cells were then lysed, and immunoblot analysis performed by using the antibodies against Akt, Erks, JNKs, or p38 kinase or the phosphospecific antibodies against their phosphorylated proteins. Antibodybound proteins were detected by chemiluminescence (ECF from Amersham Pharmacia Biotech) and analyzed using the Storm 840 scanner The cells were then irradiated with UVB (4 kJ/m 2 ) and cultured for 15 or 30 min. The cells were lysed, and the activities of PI3-K (left), Erks (top right), p38 kinase (middle right), or JNKs (bottom right) were determined as described under "Experimental Procedures." PI3P, phosphoinositol 3-monophosphate. B, the transfectant cells were treated as described above and lysed. The levels of phosphorylation and activation of Akt were assessed by immunoblotting. C, the transfectant cells were treated as described above and lysed. Akt was immunoprecipitated from the lysates using anti-Akt1/PKB␣, PH domain antibody. The activity of Akt was measured using Akt substrate peptide and [␥-32 P]ATP. Columns and bars indicate the mean Ϯ S.E. of at least three experiments.

(Molecular Dynamics).
Assay for Erk, p38 Kinase, and JNK Activities-Cells were treated with UVB (4 kJ/m 2 ) as described above. The cells were lysed in 400 l of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , 1 mm ␤-glycerophosphate, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 1 M microcystin). The lysates were sonicated and centrifuged, and the supernatant fraction was incubated with the phospho-specific Erks, p38 kinase, or JNKs antibody with gentle rocking for 6 -10 h at 4°C. Then, the protein A/G plus agarose was added, and the incubation was continued for another 4 h. The beads were washed twice with 500 l of lysis buffer and twice with 500 l of kinase buffer (25 mM Tris, pH 7.5, 5 mM ␤-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ). The kinase reactions were carried out in the presence of 200 M ATP at 30°C for 30 min using 2 g of Elk-1, ATF-2, or c-Jun as substrate for Erks, p38 kinase, or JNKs, respectively. The phosphorylated proteins were detected by immunoblotting using phospho-specific antibodies.
Akt Phosphorylation Assay-Cells were treated with UVB (4 kJ/m 2 ), lysates were prepared from the cells, and the immunoprecipitation was carried out using the phospho-specific Erk, p38 kinase, or JNKs antibody or the specific Msk1, MAPKAP-K2, or Rsk1 antibody as described above (under "Assay for Erk, p38 Kinase, and JNK Activities"). The kinase reactions were carried out at 30°C for 30 min in the presence of kinase buffer with 200 M ATP and 250 ng of inactive Akt fusion protein as substrate. The phosphorylated protein was detected by immunoblotting using phospho-specific Akt antibodies.
Radioactive Akt and Msk1 Immunoprecipitation Kinase Assay-Cells were treated with UVB (4 kJ/m 2 ) or insulin (2.5 g/ml), lysates were prepared from the cells, and the immunoprecipitation was carried out using 4 g of anti-Akt1/PKB␣ PH domain or anti-Msk1 antibody as described above. The enzyme immune complex was washed three times with 0.5 ml of lysis buffer and once with 100 l of assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerophosphate, pH 7.0, 1 mM sodium orthovanadate, 1 mM DTT). For the Akt kinase assay, the enzyme immune complex was added to 10 l of assay dilution buffer, 40 M protein kinase A inhibitor peptide, 0.4 mM Akt substrate peptide, and 1 Ci/l [␥-32 P]ATP, and for the Msk kinase assay, it was added to 20 l of assay dilution buffer, 10 l of 0.4 mM Akt substrate peptide, and 1 Ci/l [␥-32 P]ATP. The reaction was incubated for 10 min at 30°C and centrifuged, and then 30 l of the supernatant fraction was transferred onto P81 phosphocellulose paper and allowed to bind for 30 s. The P81 papers were washed three times in 0.75% phosphoric acid and then washed once in acetone, and ␥-32 P incorporation was measured by scintillation counting.
Nonradioactive Akt Immunoprecipitation Kinase Assay-Cells were treated with UVB (4 kJ/m 2 ), lysates were prepared from the cells, and the immunoprecipitation was carried out using 4 g of anti-Akt1/PKB␣ PH domain antibody as described above. The enzyme immune complex was added to 12.5 l of assay dilution buffer, 10 l (3 g) of Bad protein, and 50 l of magnesium/ATP mixture (75 mM MgCl 2 and 500 M ATP in assay dilution buffer). The reaction was incubated for 10 min at 30°C and centrifuged, and then 20 l of the supernatant fraction was used as a sample for immunoblotting. Immunoblot analysis was performed by using the phospho-specific Bad (serine 136) antibody, and antibodybound proteins were detected by chemiluminescence (ECF from Amersham Pharmacia Biotech) and analyzed using the Storm 840 scanner (Molecular Dynamics). Activation of Akt by UVB-activated Msk-Cells were treated with UVB (4 kJ/m 2 ), lysates were prepared from the cells, and the immunoprecipitation was carried out using 4 g of anti-Msk1 or anti-MAP-KAP-K2 antibodies as described above. The enzyme immune complex was added to 12.5 l (250 ng) of Akt in assay dilution buffer, 10 l (3 g) of Bad protein, and 50 l of magnesium/ATP mixture (75 mM MgCl 2 and 500 M ATP in assay dilution buffer). The reaction was incubated for 30 min at 30°C and centrifuged, and then 20 l of the supernatant fraction was used as a sample of immunoblotting. Immunoblot analysis was performed by using the phospho-specific Bad (serine 136) antibody. Antibody-bound proteins were detected by chemiluminescence (ECF from Amersham Pharmacia Biotech) and analyzed using the Storm 840 scanner (Molecular Dynamics).

Induction of Akt Phosphorylation by UVB Irradiation-Be-
cause the phosphorylation of threonine 308 and serine 473 is a prerequisite for the catalytic activity of Akt (19,(33)(34)(35), we first investigated whether exposure of cells to UVB irradiation results in phosphorylation of Akt. As shown in Fig. 1, phosphorylation of Akt at both threonine 308 and serine 473 occurred 5 min after cells were exposed to UVB irradiation (4 kJ/m 2 ) and reached a maximum at 30 min.
Inhibition of UVB-induced Phosphorylation and Activation of Akt by Pretreatment of Cells with LY 294002, PD 98059, or SB 202190 -Akt is an established downstream target of PI3-K that is activated following stimulation of cells with growth factors (11,12,(15)(16)(17). UVB-induced phosphorylation and activation of Akt were blocked by pretreatment with LY 294002, a PI3-K inhibitor (Fig. 2), showing that UVB-induced Akt activation is mediated through the PI3-K pathway. UV irradiation also leads to activation of the MAP kinase superfamily, composed of Erks, JNKs, and p38 kinase (9, 40 -46). Therefore, we investigated the possible role of the MAP kinase family in mediating this process. To do this, we examined the influence of specific chemical inhibitors of MAP kinases on UVB-induced phosphorylation and activation of Akt. Interestingly, UVBinduced phosphorylation and activation of Akt were impaired by pretreatment of cells with PD 98059, a specific MAP kinase/ Erk kinase 1 inhibitor, and/or SB 202190, a specific p38 kinase inhibitor (Fig. 2). These data suggest that both Erks and p38 kinase may be involved, in addition to the PI3-K pathway, in UVB-induced Akt activation.

UVB-induced Phosphorylation and Activation of Akt in Cells Expressing a Dominant Negative Mutant of p85, Erk2, p38
Kinase, or JNK1-To directly investigate the role of PI3-K and the MAP kinase family in UVB-induced Akt activation, we used dominant negative mutant transfectants of Erk2 (DN-Erk2) (44), p38 kinase (DN-p38) (56), JNK1 (DN-JNK1) (55), or PI3-K p85 subunit (DN-p85) (57). Overexpression of DN-Erk2, DN-p38, or DN-JNK1 specifically blocked UVB-induced Erk, p38 kinase, or JNK activities, respectively (Fig. 3A, right panel) (44,55,56). We also confirmed that expression of DN-p85 inhibits UVB-induced PI3-K activity (Fig. 3A, left panel). UVBinduced phosphorylation and activation of Akt was blocked by the expression of DN-p85 (Fig. 3, B and C). The expression of DN-Erk2 or DN-p38 attenuated UVB-induced phosphorylation and activation of Akt, whereas its phosphorylation and activation were not inhibited in cells expressing DN-JNK1 (Fig. 3, B and C). The difference in the strength of inhibition observed between the chemical inhibitor and DN-p38 cells for Akt activation may be caused by an inferior dominant negative effect due to loss of the transfection efficacy of the cDNA.
Erk Activation by UVB Is Mediated by PI3-K-Several studies have shown that PI3-K inhibitors repress Erk activation (58,59). We assessed the effect of LY 294002 on UVB-induced phosphorylation of MAP kinases. The results showed that LY 294002 blocked UVB-induced Erk phosphorylation but not JNK or p38 kinase phosphorylation (Fig. 4). Moreover, cells expressing DN-p85 also blocked only UVB-induced Erk phosphorylation (Fig. 4). Thus, these results indicated that in UVB signaling, the Erk pathway is mediated by PI3-K.
Erks and p38 Kinase Regulate the Activation of Akt by UVB through Msk1-To test whether Erks and p38 kinase are direct mediators of UVB-induced Akt activation, we carried out immune complex phosphorylation assays of UVB-activated Erks, p38 kinase, or JNKs, which were immunoprecipitated from cells exposed to UVB, using an inactive Akt fusion protein as the substrate. In these experiments, the Akt protein was not phosphorylated by UVB-activated Erks, p38 kinase, or JNKs (Fig. 5A). Among the substrates of MAP kinases are members of a second family of protein-serine/threonine kinases, including Rsks, Msks, and MAPKAP-Ks (MAPKAP-K2 and MAP-KAP-K3). Rsks are activated by Erks (60), Msks are activated by Erks and p38 kinase (53,61), and MAPKAP-Ks are activated by p38 kinase (62,63). These protein-serine/threonine kinases (Rsk1, Msk1, and MAPKAP-K2) are also activated in JB6 Cl 41 cells by UVB irradiation (data not shown and Figs. 7 and 8). We therefore determined whether Akt is activated by these protein-serine/threonine kinases. As shown in Fig. 5B,  (64). Using the specific substrate Bad with the Akt fusion protein, UVB-activated Msk1 (but not UVB-activated MAPKAP-K2) was also observed to induce the activation of Akt (Fig. 6). Thus, phosphorylation of both sites may be necessary for Akt activation. These results suggest that Akt is, at least in part, phosphorylated via Msk1.
UVB-induced Msk1 activity was at a maximum at 30 min, corresponding to UVB-induced Akt phosphorylation (Fig. 7A), and the activity was inhibited by pretreatment of cells with PD 98059 and/or SB 202190 (Fig. 7B). Furthermore, LY 294002, which blocks UVB-induced Erk activation, also partially impaired UVB-induced Msk1 activity (Fig. 7B). Thus, Erk and p38 kinase pathways were confirmed to mediate UVB-induced Msk1 activation.
Insulin-induced Akt Activation Is Independently Stimulated with Msk1-Akt is known to be activated by treatment with insulin or growth factor (11,12). To confirm whether insulininduced Akt phosphorylation is also mediated through Msk1, we investigated the involvement of Msk1 on insulin-induced Akt1 phosphorylation. Phosphorylation of Akt was induced by the treatment with insulin for 5 min (Fig. 8A). Msk1 was only weakly activated by the treatment with insulin and the activation was a maximum by the treatment with insulin for 30 min (Fig. 8B). Insulin-induced Msk1 activation was inhibited by the pretreatment with PD 98059 (Fig. 9C), whereas insulininduced phosphorylation and activation of Akt did not markedly change by PD 98059 (Fig. 9, A and B). On the other hand, insulin-induced phosphorylation and activation of Akt were completely blocked by the pretreatment with LY 294002 (Fig.  9, A and B). These results suggested that Msk1 does not mediate insulin-induced Akt activation.

Msk1
Mediates UVB-induced Phosphorylation and Activation of Akt-To test the requirement for Msk1 in UVB-induced Akt activation, we established a stable transfectant with a Msk1 C-terminal kinase-dead mutant (53). The expression of a Msk1 C-terminal kinase-dead mutant (Msk1 C-dead) inhibited UVB-induced Msk1 activity (Fig. 10A). UVB-induced phosphorylation and activation of Akt was attenuated in the Msk1 C-terminal kinase-dead mutant transfectant cells (Fig. 10, B and C). These results further supported the hypothesis that Msk1 mediates UVB-induced Akt activation.

DISCUSSION
Akt is a multifunctional kinase that appears to have numerous substrates in vivo, and it promotes cell survival by inhibiting apoptosis in response to growth factors and cytokines (11, 12, 15-17, 19, 65). Several studies have shown that acute metabolic effects, such as protein synthesis and glycogenesis, and cell cycle regulation are also associated with activation of Akt (18,19). The activation of Akt requires phosphorylation at two sites, threonine 308 and serine 473. A constitutively active kinase, PDK-1 (34,35), has been shown to phosphorylate threonine 308, but the kinase (PDK-2) responsible for phosphorylating serine 473 has not been identified. In this study, we demonstrated that Akt is phosphorylated at both threonine 308 and serine 473 and activated in response to UVB irradiation. Furthermore, we found that Erk-and p38 kinase-dependent Msk1 activation, in addition to the PI3-K/PDK-1 pathway, is required for the UVB-induced Akt activation.
Exposure of cells to UV irradiation elicits a complex set of acute cellular responses called "UV responses." The initial signal triggering the UV response is in large part independent of DNA damage, but it instead appears to be mediated by a membrane-associated component of the Ras pathway and activation of MAP kinases (7-10, 66, 67). Upstream signaling path- ways leading to Akt activation include PI3-K and Ras (11)(12)(13), and Akt is believed to be a multifunctional mediator of PI3-Kdependent signaling (11-13, 15-17, 19). UVB-induced Akt activation was also mediated by the PI3-K pathway. Akt has been shown to be overexpressed in ovarian, prostate, breast, and pancreatic cancers in humans and is associated with a poor prognosis and increased tumorigenicity (21)(22)(23). Mutation or down-regulation of PTEN, which directly antagonizes PI3-K, is frequently observed in a number of cancers (24,25), and the mutation is associated with Akt activation (26 -28). In addition to an inhibitory effect on apoptosis, Akt is known to regulate cell cycle progression (18,19). Therefore, Akt activation induced by UVB may be implicated in tumorigenesis by its effects on growth progression and promotion of survival of cells damaged by UVB irradiation. In the present study, we found that Erks and p38 kinase, but not JNKs, mediate Akt activation. PI3-K inhibitors repressed Erk activation in several cell types after various modes of stimulation, as previously described (58,59,68). Overexpression of DN-p85 also impaired UVB-induced Erk phosphorylation, although the blocking of Erk activation has been suggested to be independent of PI3-K activity (69,70). Thus, in the present study, Erk activation by UVB was shown to be mediated via PI3-K.
Erks are involved in survival signaling in response to a variety of growth factors (47,48,71), whereas activation of JNKs or p38 kinase is suggested to play decisive roles in the control of cell death (71). The activation of JNKs and p38 kinase and overexpression of MAP kinase kinase 6, an upstream kinase of p38 kinase, also have been reported to protect cells from apoptosis (72,73). However, little direct evidence has been obtained to show that the MAP kinase family regulates Akt. Therefore, the results of our study indicating that Erks and p38 kinase mediate Akt activation suggest a novel role for MAP kinases in signal transduction. In the present study, members of the MAP kinase family did not directly phosphorylate Akt. Activated MAP kinases are translocated to the nucleus, where they phosphorylate several different transcription factors (41, 53, 74 -79). In addition to phosphorylating nuclear proteins, several cytoplasmic proteins (e.g. Rsks and Msks), phospholipase A2, and the epidermal growth factor receptor have been shown to be substrates for Erks (41,53,60,61,74,80), and p38 kinase has been shown to phosphorylate cytoplasmic proteins (e.g. MAPKAP-Ks and Msks) (53,62,63). In contrast to Erks and p38 kinase, which appear to have FIG. 9. Insulin-induced phosphorylation and activation of Akt is not mediated through Msk1. A, JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cell were then pretreated with LY 294002, PD 98059, or SB 202190 for 1 h at the indicated concentrations. The cells were treated with insulin (2.5 g/ml) for 5 min. The cells were lysed, and the phosphorylation levels were assessed by immunoblotting. B, lysates were prepared from the cells treated as described above, and Akt was immunoprecipitated using anti-Akt/PKB␣, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [␥-32 P]ATP. Columns and bars indicate the mean Ϯ S.E. of at least three experiments. C, lysates were prepared from the cells treated as described above, and Msk1 was immunoprecipitated using anti-Msk1 antibody.  The cells were then irradiated with UVB (4 kJ/m 2 ) and cultured for 30 min. Lysates were prepared from the cells, and Msk1 was immunoprecipitated using anti-Msk1 antibody. Msk1 activity was determined as described under "Experimental Procedures." Columns and bars indicate the mean Ϯ S.E. of at least three experiments. B, the transfectant cells were treated as described above and lysed. The levels of phosphorylation and activation of Akt were assessed by immunoblotting. C, the transfectant cells were treated as described above and lysed. Akt was immunoprecipitated from the lysates using anti-Akt1/PKB␣, PH domain antibody. The activity of Akt was determined as described under "Experimental Procedures." Columns and bars indicate the mean Ϯ S.E. of at least three experiments. stimulated Akt activation, and overexpression of a Msk Cterminal kinase-dead mutant inhibited UVB-induced Akt activation. Although MAPKAP-K2 has been suggested to function as PDK-2 for Akt in human neutrophils (38), UVB-activated MAPKAP-K2 did not stimulate Akt activation. A previous report suggested that phosphorylation of Akt at serine 473 may occur through an autophosphorylation mechanism following PDK-1-dependent phosphorylation of Akt at threonine 308 (37). Thus, the activation of Akt is suggested to require its phosphorylation at threonine 308 or at both threonine 308 and serine 473. Our results demonstrated that Erks and p38 kinase mediate UVB-induced Akt activation via Msk1. On the other hand, Akt activation by Msk1 may be specific in the UVB response, because PD 98059, which inhibited insulin-induced Msk1 activation, did not block insulin-induced phosphorylation and activation of Akt. Although combined addition of PD 98059 and SB 202190 completely inhibited UVB-induced Msk1 activation, the combined addition partially blocked UVB-induced Akt activation. In addition, only partial inhibition of UVBinduced Msk1 activity was observed in comparison to the complete inhibition of Akt activity by a PI3-K inhibitor, suggesting that other kinase pathways, such as the PI3-K/PDK-1 pathway, are also involved in Akt activation in UVB signaling.
Taken together, the data demonstrate that Akt activation induced by UVB irradiation is mediated by the signaling of Erks and p38 kinase and their downstream protein-serine/ threonine kinase, Msk1. Because Akt is a multifunctional kinase related to cell survival, cell cycle regulation, and tumorigenesis, these results provide important information regarding signal transduction and mechanisms associated with UV responses and tumor promotion.