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Activation of SGK1 by HGF, Rac1 and integrin-mediated cell adhesion in MDCK cells: PI-3K-dependent and -independent pathways

Candace Shelly and Roman Herrera*

Department of Cell Biology, Global Research and Development, Ann Arbor Laboratories, Pfizer, Co, Ann Arbor, MI 48105, USA



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  		Fig. 1. Activation of SGK1 by HGF is wortmanin dependent. (A) Time course of SGK1 activation by HGF. MDCK cells were transiently transfected with HA-SGK1, stimulated with HGF and at the indicated times, cells were harvested and HA-SGK1 activity was measured as described in the Materials and Methods. The lower insert depicts the amount of HA-SGK1 present in the assay at the indicated times. (B) Time course activation of AKT by HGF. Cell extracts prepared as in (A) were separated in a SDS-PAGE and analyzed for the presence of phosphorylated AKT (T473)(pAKT) or total AKT as described in the Materials and Methods. (C) HGF activation of HA-SGK1 or AKT is blocked by wortmanin. MDCK cells were transfected with HA-SGK1, stimulated with HGF for 10 minutes in the presence or absence (DMSO) of wortmanin, and the kinase activity associated with HA-SGK1 was measured as in (A). The lower insert depicts the amount of phosphorylated AKT (T473)(pAKT), total AKT and HA-SGK1 present under the conditions described. Despite HGF-treated extract being under-loaded in the AKT gel, it contains detectable levels of pAKT.

 


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  		Fig. 2. Activation of HA-SGK1 by Rac1V12 is wortmanin independent. (A) MDCK cells were co-transfected with HA-SGK1 and either Rac1V12 (lane 1), RhoL63 (lane 2), Rap1V12 (lane3) or HA-SGK alone (lane 4), and the kinase activity associated with HA-SGK1 was assayed as described in the Materials and Methods. The results are presented as the fold increase in activation over the activity obtained in the absence of the small GTP-binding proteins. The lower insert depicts the amount of HA-SGK1 present in the assay. (B) Dose-dependent activation of HA-SGK1 by Rac1V12. MDCK cells were cotransfected with HA-SGK1, and the indicated amounts of Rac1V12 and the kinase activity was assayed as described in the Materials and Methods. The lower insert depicts the amount of HA-SGK1 present in the assay. (C) The effects of wortmanin on RacV12 activation of HA-SGK1. HA-SGK1 was cotransfected with Rac1V12, and cell extracts were prepared from wortmanin or vehicle-treated cells as described in the legend to Fig. 1C. The lower insert depicts the amount of HA-SGK1 present in the assay. (D) The HGF-mediated activation of HA-SGK1 is not blocked by RacN17. MDCK cells were cotransfected with HA-SGK and an empty vector or a vector containing myc-tagged RacN17. The amount of SGK in the kinase assay is depicted in the HA-SGK blot. Verification of myc-RacN17 expression is shown in the lower blot. The kinase activity was carried out as described in the legend to Fig. 2A.

 


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  		Fig. 3. SGK is not regulated by cAMP. Top, MDCK cells were transfected with wild-type, Thr369A mutant or S422D mutant SGK and cells were stimulated with 1 mM cAMP analog before SGK1 activity was measured. Kinase assay was carried out as described in the Materials and Methods. Bottom. The cell extracts were probed for phosphorylated CREB to verify cAMP stimulation and probed with anti-HA to determine the amount of HA-SGK in the kinase assay.

 


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  		Fig. 4. SGK activation does not correlate with membrane translocation. (A) GFP-SGK1 is a kinase-active protein. MDCK cells were transfected with empty vector (GFP) or GFP-SGK1 (SGKwt) and stimulated by HGF for 15 minutes. Kinase activity was determined as described in the Materials and Methods. The lower insert depicts the amount of GFP-SGK present in the assay. (B) MDCK cells were transfected with GFP, GFP-SGK or GFP-PH/AKT (the pleckstrin homology domain of AKT) and stimulated with HGF for 15 minutes. A parallel cotransfection experiment between the above vectors and RacV12 is also shown. After fixing the cells, the location of the GFP derivatives was determined by confocal microscopy as described in the Materials and Methods.

 


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  		Fig. 5. Adhesion of MDCK cells to fibronectin activates HA-SGK1 in a wortmanin-insensitive manner. (A) Time course of HA-SGK1 activation upon cell adhesion to fibronectin-coated plates. Transfected MDCK cells were allowed to bind to fibronectin-coated plates, and HA-SGK1 activity was measured at the indicated times as described in the Materials and Methods. The insert depicts the level of HA-SGK1 present in the assay. (B) Adhesion-dependent activation of AKT. Cell extracts prepared as in (A) and were analyzed for the presence of phosphorylated AKT (pAKT). (C) Adhesion-mediated activation of HA-SGK1 is not blocked by wortmanin. HA-SGK1 activation by adhesion to fibronectin coated plates was studied in the presence or absence of wortmanin. After 30 minutes of adhesion to fibronectin, SGK1 activity was measured as in (A). The insert depicts the level of HA-SGK1 present in the assay. (D) Adhesion-dependent activation of AKT is blocked by wortmanin. Cells extracts were prepared as in (C) and were analyzed for the presence of phosphorylated AKT (T473) (pAKT).

 


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  		Fig. 6. Adhesion-mediated activation of HA-SGK1 is not blocked by Rac1N17. MDCK cells were cotransfected with HA-SGK1 and myc-Rac1V12 or myc-Rac1N17, allowed to adhere to fibronectin and the activation of HA-SGK1 was measured as described in Fig. 1. The lower insert depicts the levels of HA-SGK1, Rac1N17 and Rac1V12 present in the assay.

 


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  		Fig. 7. Deattachment-induced FKHRL1 dephosphorylation is prevented by activated SGK1. MDCK cells were transfected with activated SGK1(S/D) (lanes 1, 2, 5 and 6) or empty vector (pKH3) (lanes 3, 4, 7 and 8) and serum starved overnight. Cells were deattached by trpysin incubation and kept in suspension for 1 hour (1 hour) or lysed immediately (0 hours). Duplicated cell extracts were prepared and analyzed for the presence of phosphorylated FKRHL1 (T32) (top panel), total FKRHL1 (middle panel) or for the presence of HA-SGK1 (bottom panel)

 

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