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VASCULAR BIOLOGY

Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells

Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

Submitted 13 October 2003 ; accepted in final form 5 April 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Angiotensin II activates a variety of signaling pathways in vascular smooth muscle cells (VSMCs), including the MAPKs and Akt, both of which are required for hypertrophy. However, little is known about the relationship between these kinases or about the upstream activators of Akt. In this study, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive kinase p38 MAPK and its substrate MAPKAPK-2 mediate Akt activation in VSMCs. In unstimulated VSMCs, Akt and p38 MAPK are constitutively associated and remain so after angiotensin II stimulation. Inhibition of p38 MAPK activity with SB-203580 dose-dependently inhibits Akt phosphorylation on Ser⁴⁷³, but not Thr³⁰⁸. Angiotensin II-induced phosphorylation of MAPKAPK-2 is also attenuated by SB-203580, as well as by inhibitors of ROS. In addition, angiotensin II stimulates the association of MAPKAPK-2 with the Akt-p38 MAPK complex, and an in vitro kinase assay shows that MAPKAPK-2 immunoprecipitates of VSMC lysates phosphorylate recombinant Akt in an angiotensin II-inducible manner. Finally, intracellular delivery of a MAPKAPK-2 peptide inhibitor blocks Akt phosphorylation on Ser⁴⁷³. These results suggest that the p38 MAPK-MAPKAPK-2 pathway mediates Akt activation by angiotensin II in these cells by recruiting active MAPKAPK-2 to a signaling complex that includes both Akt and p38 MAPK. Through this mechanism, p38 MAPK confers ROS sensitivity to Akt and facilitates downstream signaling. These results provide evidence for a novel signaling complex that may help to spatially organize hypertrophy-related, ROS-sensitive signaling in VSMCs.

mitogen-activated protein kinase; reactive oxygen species

Although it has been clearly shown that activation of Akt requires phosphatidylinositol 3-kinase (PI3-K) activity and NAD(P)H oxidase-derived production of ROS (6, 26), the full complement of upstream mediators of Akt activation by ANG II is not yet fully understood. Akt activation requires phosphorylation of two amino acid residues by upstream kinases: threonine 308 (Thr³⁰⁸) by 3-phosphoinositide-dependent protein kinase (PDK)-1 and serine 473 (Ser⁴⁷³) by a "so-called" PDK2, whose identity is controversial (4). To date, five kinases have been proposed as candidates for PDK2: Akt itself, PDK1, integrin-linked kinase (ILK), conventional PKC isoforms, and MAPKAPK-2 (15).

In VSMCs, the shared ROS sensitivity of p38 MAPK and Akt suggests that they are in the same signaling axis leading to hypertrophy. In the present study, we tested the hypothesis that p38 MAPK mediates Akt activation via MAPKAPK-2 because MAPKAPK-2 is a known substrate of p38 MAPK (19) and phosphorylates Akt in vitro (1). We found not only that p38 MAPK is required for ANG II-induced Akt activation, but also that ANG II stimulates the recruitment of MAPKAPK-2 to p38 MAPK-Akt complexes, where MAPKAPK-2 can phosphorylate Akt on Ser⁴⁷³. Thus the redox sensitivity of Akt is conferred via the upstream ROS-sensitive kinase p38 MAPK. These results provide evidence for a novel signaling complex that integrates diverse upstream signals contributing to VSMC hypertrophy.

	MATERIALS AND METHODS

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Materials. Anti-phospho-Akt1/PKB- (Thr³⁰⁸) was obtained from Upstate Biotechnology (Lake Placid, NY). Phospho-MAPKAPK-2 (Thr²²²) antibody, phospho-Akt (Ser⁴⁷³), Akt antibody, phospho-p38 MAPK antibody, and p38 MAPK antibody were from Cell Signaling Technology (Beverly, MA). MAPKAPK-2 antibody was from Stressgen Biotechnologies (Victoria, BC). Protein A/G Plus-agarose was from Santa Cruz Biotechnology (Santa Cruz, CA). SB-203580, PD-98059, PD-169316, and MAPKAPK-2 inhibitor were from Calbiochem (San Diego, CA). Enhanced chemiluminescence reagents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Chariot delivery reagent was from Active Motif (Carlsbad, CA). All other chemicals and reagents, including DMEM with 25 mM HEPES and 4.5 g/l glucose, were from Sigma (St. Louis, MO).

Cell culture. VSMCs were isolated from male Harlan Sprague-Dawley rat thoracic aortas by enzymatic digestion, as described previously (10). Cells were grown in DMEM supplemented with 10% calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells between passages 6 and 17 were used for experiments.

For MAPKAPK-2 peptide inhibitor experiments, the MAPKAPK-2 inhibitor was first incubated with the peptide delivery agent Chariot for 30 min (1:1 vol/vol). VSMCs at 40–50% confluence were incubated in serum-free medium with the Chariot-peptide complex for 1 h at 37°C and allowed to recover in 0.1% calf serum containing DMEM for 1 h before treatment with ANG II.

Western blotting. VSMCs at 80–90% confluence in 100-mm dishes were made quiescent by incubation with DMEM containing 0.1% calf serum for 24 h. Cells were stimulated with agonist at 37°C in serum-free DMEM for the specified durations. Some cells were preincubated with various inhibitors, as indicated. After treatment, cells were lysed with 500 µl of ice-cold lysis buffer, pH 7.4 (in mM: 50 HEPES, 5 EDTA, 100 NaCl), 1% Triton X-100, protease inhibitors (10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin), and phosphatase inhibitors (in mM: 50 sodium fluoride, 1 sodium orthovanadate, 10 sodium pyrophosphate, 0.001 microcystin). Solubilized proteins were centrifuged at 27,000 g at 4°C for 15 min, and supernatant protein was quantified by the Bradford assay. Proteins were separated by using 9% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked at room temperature with PBS containing 5% nonfat dry milk and 0.1% Tween 20 for 1 h. Blots were incubated with the indicated primary antibodies at 4°C for 16 h. After incubation with secondary antibodies (horseradish peroxidase conjugated), proteins were detected by enhanced chemiluminescence. Band intensity was quantified by densitometry of immunoblots by using NIH Image, version 1.61.

Immunoprecipitation. For immunoprecipitation, VSMCs were lysed as described above, cell lysates (1 mg) were incubated with the indicated antibodies overnight, and the immunocomplex was collected with 20 µl of protein A/G-agarose beads for 2 h at 4°C with gentle rocking. The beads were washed four times with 500-µl lysis buffer and boiled in 1x SDS sample buffer.

MAPKAPK-2 activity assay. VSMC lysates were prepared and subjected to immunoprecipitation with anti-MAPKAPK-2 antibody, as described above. The beads were washed 3x with lysis buffer and once with kinase buffer [20 mM Tris (pH 7.4), 10 mM MnCl₂, 1 mM dithiothreitol]. The kinase reaction was carried out by incubating the beads in 30-µl kinase buffer containing 5 µM ATP, 5 mM MgCl₂, and 200 µM recombinant Akt for 30 min at 30°C. The kinase reaction was terminated by boiling samples in SDS sample buffer. Proteins were separated on a 9% SDS-polyacrylamide gel. Akt phosphorylated at Ser⁴⁷³ was detected by immunoblotting with phosphorylation site-specific antibody followed by densitometry by using NIH Image, version 1.61.

Statistical analysis. Results are expressed as means ± SE. Statistical significance was assessed by ANOVA, followed by Bonferroni's test. A value of P < 0.05 was considered to be statistically significant.

	RESULTS

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Time course of p38 MAPK and Akt phosphorylation. Our laboratory has previously shown that ANG II activates both Akt and p38 MAPK in VSMCs (25, 26). To examine in more detail the relative time courses of phosphorylation of these two kinases, we performed Western analysis with phospho-specific antibodies. As shown in Fig. 1, ANG II induced a rapid phosphorylation of p38 MAPK, which was detected as early as 1 min and peaked at 5 min. Similarly, Akt was phosphorylated on Ser⁴⁷³ by ANG II, beginning at 2 min and reaching a maximum at 5 min. These time courses are consistent with the hypothesis that p38 MAPK is upstream of Akt.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Time course of p38 MAPK and Akt phosphorylation by ANG II. Vascular smooth muscle cells (VSMCs) were stimulated with 100 nM ANG II for the indicated times and lysed, and proteins were separated by SDS-PAGE. Western analysis was performed by using phospho-specific antibodies to detect phosphorylated p38 MAPK (p-p38 MAPK) and Akt phosphorylated on Ser473 [p-Akt (pS473)]. Blots for total protein are also shown (p38 MAPK or Akt). Each blot is representative of 3–4 experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Effect of p38 MAPK inhibition on phosphorylation of Akt. VSMCs were pretreated with vehicle or SB-203580 (1 or 10 µM) for 30 min and then stimulated with 100 nM ANG II for 5 min. Western analysis was performed by using site- and phospho-specific Akt antibodies against either Ser473 [p-Akt (pS473); A] or Thr308 [p-Akt (pT308); B] or total Akt antibodies (bottom blots). Bar graphs represent averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation observed in unstimulated cells was defined as 1.0. Values are means ± SE for 4 independent experiments. *P < 0.05 vs. unstimulated cells; #P < 0.05 vs. ANG II alone; **P < 0.01 vs. ANG II alone.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Coimmunoprecipitation of Akt and p38 MAPK. VSMCs were stimulated with 100 nM ANG II for the indicated time, lysed, and immunoprecipitated with anti-Akt antibody. Proteins were separated by SDS-PAGE, and Western analysis was performed with the use of anti-p38 MAPK antibody. This blot is representative of 2 independent experiments. B, buffer blank; IP, immunoprecipitation; IB, immunoblot.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Time course of MAPKAPK-2 phosphorylation by ANG II. VSMCs were stimulated with 100 nM ANG II for the indicated times and lysed, and proteins were separated by SDS-PAGE. Western analysis was performed with the use of phospho-specific antibody to detect MAPKAPK-2 phosphorylated on Thr222 (p-MAPKAPK-2). Blot for total protein is also shown (MAPKAPK-2). Bar graph represents averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation observed in unstimulated cells at time 0 was defined as 1.0. Values are means ± SE for 4 independent experiments. *P < 0.05 vs. time 0.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Effect of p38 MAPK inhibition on phosphorylation of MAPKAPK-2. VSMCs were pretreated with vehicle or SB-203580 (1 or 10 µM) for 30 min and then stimulated with 100 nM ANG II for 5 min. Western analysis was performed with the use of anti-phospho-specific antibodies against Thr222 of MAPKAPK-2 (top blot) or total MAPKAPK-2 antibodies (bottom blot). Bar graph represents averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation observed in unstimulated cells was defined as 1.0. Values are means ± SE for 6 independent experiments. *P < 0.05 vs. unstimulated cells; **P < 0.01 vs. ANG II alone.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Reactive oxygen species (ROS) sensitivity of ANG II-induced MAPKAPK-2 phosphorylation. VSMCs were pretreated with 20 mM N-acetylcysteine or 10 µM diphenylene iodonium for 30 min before addition of 100 nM ANG II for 5 min. Cells were lysed and prepared for Western analysis with the use of phospho-specific antibody to detect MAPKAPK-2 phosphorylated on Thr222 (p-MAPKAPK-2) or total MAPKAPK-2 (top). Bar graph represents averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation observed in unstimulated cells was defined as 1.0. Values are means ± SE for 4 independent experiments. *P < 0.05 vs. control; **P < 0.05 vs. ANG II alone.

View larger version (46K): [in this window] [in a new window]   Fig. 7. Coimmunoprecipitation of Akt and MAPKAPK-2. VSMCs were stimulated with 100 nM ANG II for 2 min, lysed, and immunoprecipitated with anti-MAPKAPK-2 antibody. Proteins were separated by SDS-PAGE, and Western analysis was performed with the use of anti-Akt or anti-MAPKAPK-2 antibody. Bar graph represents averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation observed in unstimulated cells was defined as 1.0. Values are means ± SE for 3 independent experiments. *P < 0.05 vs. control.

View larger version (38K): [in this window] [in a new window]   Fig. 8. MAPKAPK-2 in vitro kinase assay for Akt. VSMCs were stimulated with 100 nM ANG II for 5 min, lysed, and immunoprecipitated with anti-MAPKAPK-2 antibody. An in vitro kinase assay was performed with the immunocomplex with the use of recombinant Akt as a substrate. Proteins were separated by SDS-PAGE, and Western analysis was performed with the use of site- and phospho-specific Akt antibody against Ser473 [p-Akt (pS473)]. Bar graph represents averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation observed in unstimulated cells was defined as 1.0. Values are means ± SE for 3 independent experiments. *P < 0.05 vs. control.

View larger version (36K): [in this window] [in a new window]   Fig. 9. Inhibition of ANG II-induced Akt phosphorylation by a peptide MAPKAPK-2 inhibitor. VSMCs were pretreated with the MAPKAPK-2 inhibitor peptide at the indicated amount for 1 h before addition of 100 nM ANG II for 5 min, as indicated in MATERIALS AND METHODS. Cells were lysed and prepared for Western analysis with the use of anti-phospho-Ser473 Akt antibody or total Akt antibody (top). Bar graph represents averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation observed in unstimulated cells was defined as 1.0. Values are means ± SE for 4 independent experiments. *P < 0.01 vs. control; **P < 0.01 vs. ANG II alone.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Candidates for PDK2 other than MAPKAPK-2 include Akt itself, PDK1, ILK, and conventional PKC isoforms. In vitro evidence exists supporting most of these kinases, but their in vivo role seems to be cell-type specific. Although Akt can autophosphorylate on Ser⁴⁷³ in vitro (23), kinase-inactive Akt can still be phosphorylated at Ser⁴⁷³ in human embryonic kidney (HEK)-293 cells (1), calling into question the importance of autophosphorylation in vivo. PDK1 also phosphorylates Ser⁴⁷³ in vitro (2); however, in PDK1^–/– embryonic stem cells, Akt is still phosphorylated at Ser⁴⁷³ in response to epidermal growth factor (27), suggesting that other mechanisms exist. ILK is a viable candidate because depletion of ILK in HEK-293 cells with the use of siRNA almost completely inhibits Ser⁴⁷³ phosphorylation (24), although it is presently unknown whether ILK exerts these effects directly or via an intermediate kinase. Finally, in platelets, activation of PKC can phosphorylate Akt on Ser⁴⁷³ in a PI3-K-independent manner (14). This is unlikely to be the case in VSMCs, however, because ANG II-induced activation of Akt is PI3-K-dependent (26), as it is for most other agonists and cell types (15).

The data presented here strongly suggest that the p38 MAPK-MAPKAPK-2 pathway regulates phosphorylation of Akt on Ser⁴⁷³ in ANG II-stimulated VSMCs. MAPKAPK-2 is a known substrate of p38 MAPK that is activated in response to cellular stresses (19). It has been shown to phosphorylate 27-kDa heat shock protein (21), the light chain of myosin II (12), and vimentin (5) and to be involved in cytoskeletal reorganization and migration (13). Furthermore, MAPKAPK-2 phosphorylates Akt on Ser⁴⁷³ in vitro (1), and a peptide inhibitor of MAPKAPK-2 blocks Ser⁴⁷³ phosphorylation in neutrophils stimulated with f-Met-Leu-Phe (18). Our data extend these in vitro observations to VSMCs and provide a mechanism by which the activation of MAPKAPK-2 and its interaction with Akt occur. We found that ANG II stimulates a dynamic association of MAPKAPK-2 with the constitutive Akt-p38 MAPK complex (Figs. 2 and 7). Moreover, MAPKAPK-2 immunocomplexes phosphorylate Ser⁴⁷³ on recombinant Akt in an ANG II-inducible manner (Fig. 8), and a MAPKAPK-2 inhibitor blocks Akt phosphorylation in intact cells (Fig. 9). Because inhibition of p38 MAPK attenuates both MAPKAPK-2 and Ser⁴⁷³ Akt phosphorylation (Figs. 3B and 5), these data suggest that p38 MAPK initiates the interaction and activation of the other kinases. The specificity of this pathway is evident from both the lack of effect of SB-230580 on Thr³⁰⁸ Akt phosphorylation (Fig. 3A) and the inability of the ERK1/2 pathway inhibitor PD-98059 to block Ser⁴⁷³ phosphorylation. Taken together, our results indicate that p38 MAPK phosphorylates MAPKAPK-2 and recruits it to the constitutively present p38 MAPK-Akt complex, permitting phosphorylation of Akt on Ser⁴⁷³.

It is noteworthy that activation of Akt occurs on recruitment of MAPKAPK-2 to the signaling complex. There is some evidence in other cell types that this occurs in the nucleus (17), placing this complex in an ideal location to mediate gene transcription. All three proteins phosphorylate specific transcription factors, and their presence in a complex is likely to provide a coordinated regulation of gene expression. Clearly, further experiments are necessary to evaluate the functional consequences of these protein-protein interactions.

Although previous work has shown that ANG II activates p38 MAPK (16, 25), MAPKAPK-2 (16), and Akt (26) in VSMCs, the present study provides a framework with which to integrate these signaling molecules and to understand the relationship between ROS-sensitive kinases important in growth (Fig. 10). ANG II stimulates NAD(P)H oxidases to increase the production of ROS (9), which, in turn, mediates both p38 MAPK and Akt activation (25, 26). We now show that p38 MAPK is upstream of Akt (Fig. 3) and that MAPKAPK-2 serves as an intermediate, ROS-sensitive kinase in this signaling pathway. The constitutive association of p38 MAPK and Akt provides a basis for their mutual ROS sensitivity and importance in hypertrophy, and the recruitment of MAPKAPK-2 to this complex supplies the mechanism by which p38 MAPK activates Akt.

View larger version (21K): [in this window] [in a new window]   Fig. 10. Proposed signaling pathways mediating ANG II-induced Akt activation in VSMCs. Based on our laboratory's previous work and the present study, we propose that ROS-sensitive 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activation (22) phosphorylates Thr308 of Akt (data not shown). A parallel pathway is activated that involves NAD(P)H oxidase (Nox)-dependent generation of ROS, leading to activation of p38 MAPK (25). p38 MAPK exists in a complex with Akt, and activation of p38 MAPK leads to recruitment of MAPKAPK-2 to the p38 MAPK-Akt complex, its phosphorylation, and activation. MAPKAPK-2 then phosphorylates Akt on Ser473, leading to full Akt activation. p22, p22phox; PI3-K, phosphatidylinositol 3-kinase; AT1R, ANG II type 1 receptor.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-38206, HL-58000, and HL-60728; a grant from the Japan Heart Foundation and Bayer Yakuhin Research Grant Abroad (to Y. Taniyama); and American Heart Association Scientist Development Grant 0130175N (to M. Ushio-Fukai).

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. K. Griendling, Emory Univ., Division of Cardiology, 319 WMB, 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: kgriend{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, and Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15: 6541–6551, 1996.[Web of Science][Medline]

2. Balendran A, Casamayor A, Deak M, Paterson A, Gaffney P, Currie R, Downes CP, and Alessi DR. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr Biol 9: 393–404, 1999.[CrossRef][Web of Science][Medline]

3. Ben-Levy R, Leighton IA, Doza YN, Attwood P, Morrice N, Marshall CJ, and Cohen P. Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. EMBO J 14: 5920–5930, 1995.[Web of Science][Medline]

4. Chan TO and Tsichlis PN. PDK2: a complex tail in one Akt. Sci STKE 66: PE1, 2001.

5. Cheng TJ and Lai YK. Identification of mitogen-activated protein kinase-activated protein kinase-2 as a vimentin kinase activated by okadaic acid in 9L rat brain tumor cells. J Cell Biochem 71: 169–181, 1998.[CrossRef][Web of Science][Medline]

6. Eguchi S, Iwasaki H, Ueno H, Frank GD, Motley ED, Eguchi K, Marumo F, Hirata Y, and Inagami T. Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt. J Biol Chem 274: 36843–36851, 1999.[Abstract/Free Full Text]

7. Eguchi S, Matsumoto T, Motley ED, Utsunomiya H, and Inagami T. Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. Possible requirement of Gq-mediated p21ras activation coupled to a Ca²⁺/calmodulin-sensitive tyrosine kinase. J Biol Chem 271: 14169–14175, 1996.[Abstract/Free Full Text]

8. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, and Inagami T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273: 8890–8896, 1998.[Abstract/Free Full Text]

9. Griendling KK, Minieri CA, Ollerenshaw JD, and Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141–1148, 1994.[Abstract/Free Full Text]

10. Griendling KK, Taubman MB, Akers M, Mendlowitz M, and Alexander RW. Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells. J Biol Chem 266: 15498–15504, 1991.[Abstract/Free Full Text]

11. Ishida M, Marrero MB, Schieffer B, Ishida T, Bernstein KE, and Berk BC. Angiotensin II activates pp60c-src in vascular smooth muscle cells. Circ Res 77: 1053–1059, 1995.[Abstract/Free Full Text]

12. Komatsu S and Hosoya H. Phosphorylation by MAPKAP kinase 2 activates Mg²⁺-ATPase activity of myosin II. Biochem Biophys Res Commun 223: 741–745, 1996.[CrossRef][Web of Science][Medline]

13. Kotlyarov A, Yannoni Y, Fritz S, Laass K, Telliez JB, Pitman D, Lin LL, and Gaestel M. Distinct cellular functions of MK2. Mol Cell Biol 22: 4827–4835, 2002.[Abstract/Free Full Text]

14. Kroner C, Eybrechts K, and Akkerman JW. Dual regulation of platelet protein kinase B. J Biol Chem 275: 27790–27798, 2000.[Abstract/Free Full Text]

15. Leslie NR, Biondi RM, and Alessi DR. Phosphoinositide-regulated kinases and phosphoinositide phosphatases. Chem Rev 101: 2365–2380, 2001.[CrossRef][Web of Science][Medline]

16. Meloche S, Landry J, Huot J, Houle F, Marceau F, and Giasson E. p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle. Am J Physiol Heart Circ Physiol 279: H741–H751, 2000.[Abstract/Free Full Text]

17. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, and Davis RJ. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270: 7420–7426, 1995.[Abstract/Free Full Text]

18. Rane MJ, Coxon PY, Powell DW, Webster R, Klein JB, Pierce W, Ping P, and McLeish KR. p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J Biol Chem 276: 3517–3523, 2001.[Abstract/Free Full Text]

19. Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, and Nebreda AR. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78: 1027–1037, 1994.[CrossRef][Web of Science][Medline]

20. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, and Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res 91: 406–413, 2002.[Abstract/Free Full Text]

21. Stokoe D, Engel K, Campbell DG, Cohen P, and Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313: 307–313, 1992.[CrossRef][Web of Science][Medline]

22. Taniyama Y, Weber DS, Rocic P, Hilenski L, Akers ML, Park J, Hemmings BA, Alexander RW, and Griendling KK. Pyk2- and Src-dependent tyrosine phosphorylation of PDK1 regulates focal adhesions. Mol Cell Biol 23: 8019–8029, 2003.[Abstract/Free Full Text]

23. Toker A and Newton AC. Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. J Biol Chem 275: 8271–8274, 2000.[Abstract/Free Full Text]

24. Troussard AA, Mawji NM, Ong C, Mui A, St. Arnaud R, and Dedhar S. Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation. J Biol Chem 278: 22374–22378, 2003.[Abstract/Free Full Text]

25. Ushio-Fukai M, Alexander RW, Akers M, and Griendling KK. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem 273: 15022–15029, 1998.[Abstract/Free Full Text]

26. Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, and Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 274: 22699–22704, 1999.[Abstract/Free Full Text]

27. Williams MR, Arthur JS, Balendran A, van der Kaay J, Poli V, Cohen P, and Alessi DR. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr Biol 10: 439–448, 2000.[CrossRef][Web of Science][Medline]

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				A. Patzak, E. Y. Lai, M. Fahling, M. Sendeski, P. Martinka, P. B. Persson, and A. E. G. Persson Adenosine enhances long term the contractile response to angiotensin II in afferent arterioles Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2007; 293(6): R2232 - R2242. [Abstract] [Full Text] [PDF]

				P. K. Mehta and K. K. Griendling Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system Am J Physiol Cell Physiol, January 1, 2007; 292(1): C82 - C97. [Abstract] [Full Text] [PDF]

				J. H. Lee and L. Ragolia AKT phosphorylation is essential for insulin-induced relaxation of rat vascular smooth muscle cells Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1355 - C1365. [Abstract] [Full Text] [PDF]

				A. N. Lyle and K. K. Griendling Modulation of vascular smooth muscle signaling by reactive oxygen species. Physiology, August 1, 2006; 21: 269 - 280. [Abstract] [Full Text] [PDF]

				A.-M. Samuelsson, E. Bollano, R. Mobini, B.-M. Larsson, E. Omerovic, M. Fu, F. Waagstein, and A. Holmang Hyperinsulinemia: effect on cardiac mass/function, angiotensin II receptor expression, and insulin signaling pathways Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H787 - H796. [Abstract] [Full Text] [PDF]

				R. E. Clempus and K. K. Griendling Reactive oxygen species signaling in vascular smooth muscle cells Cardiovasc Res, July 15, 2006; 71(2): 216 - 225. [Abstract] [Full Text] [PDF]

				J. Suzuki, Z.-G. Jin, D. F. Meoli, T. Matoba, and B. C. Berk Cyclophilin A Is Secreted by a Vesicular Pathway in Vascular Smooth Muscle Cells Circ. Res., March 31, 2006; 98(6): 811 - 817. [Abstract] [Full Text] [PDF]

				F. Li and K. U. Malik Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2+-dependent phospholipase A2 Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2306 - H2316. [Abstract] [Full Text] [PDF]

				F. Li and K. U. Malik Angiotensin II-Induced Akt Activation through the Epidermal Growth Factor Receptor in Vascular Smooth Muscle Cells Is Mediated by Phospholipid Metabolites Derived by Activation of Phospholipase D J. Pharmacol. Exp. Ther., March 1, 2005; 312(3): 1043 - 1054. [Abstract] [Full Text] [PDF]

				R.M. Touyz, G. Yao, M.T. Quinn, P.J. Pagano, and E.L. Schiffrin p47phox Associates With the Cytoskeleton Through Cortactin in Human Vascular Smooth Muscle Cells: Role in NAD(P)H Oxidase Regulation by Angiotensin II Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 512 - 518. [Abstract] [Full Text] [PDF]

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