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. Author manuscript; available in PMC: 2013 May 31.
Published in final edited form as: J Biol Chem. 2000 Jun 9;275(23):17838–17847. doi: 10.1074/jbc.M909735199

Fig. 6. Selective activation of the SAPK pathway by the long form of Gene 33: Requirement for the C-terminal half of Gene 33 consisting of the 14-3-3-binding, PDZ-binding, and AH domains (the short form of Gene 33 cannot activate the SAPKs).

Fig. 6

A, activation of MAPK pathways by Gene 33. 293 cells were transfected with HA-SAPK, ERK1, or p38 as indicated plus vector or FLAG-Gene 33. As indicated, FLAG-V12-Cdc42Hs served as a positive control for SAPK activation, FLAG-V12-Ha-Ras was a positive control for ERK1, and untagged apoptosis signal-regulating kinase-1 was a positive control for p38. HA-MAPKs were immunoprecipitated with anti-HA and assayed with GST-c-Jun (SAPK), myelin basic protein (MBP, ERK assays), or GST-activating transcription factor-2 (p38) (right panels). In the left panels, crude lysates were probed with the cognate antibodies to determine expression of the transfected constructs. B, schematic diagram of Gene 33 truncation mutants. wt, wild type; BD, binding domain; C, activation of SAPK by Gene 33 deletion constructs alone or in combination with V12-Cdc42Hs. 293 cells were cotransfected with HA-SAPK and the indicated FLAG-tagged Gene 33 and/or Cdc42Hs constructs. HA-SAPK was immunoprecipitated and assayed for GST-c-Jun kinase (top panel). Crude lysates were probed with anti-HA to detect SAPK levels (middle panel) or anti-FLAG to detect Gene 33 and Cdc42Hs levels (bottom panel). D, the short form of Gene 33 cannot activate coexpressed SAPK. The top diagram shows the structure of the long and short forms of Gene 33 (Gene 33 L and Gene 33 S, respectively). The bottom panel indicates the effect of these Gene 33 constructs on the activity of coexpressed SAPK. 293 cells were cotransfected with GST-SAPK and the indicated FLAG-tagged Gene 33 and/or V12-Cdc42Hs constructs. GST-SAPK was isolated on GSH-agarose and assayed for GST-c-Jun kinase (top panel). Crude lysates were probed with anti-GST to detect SAPK levels (middle panel) or anti-FLAG to detect Gene 33 and Cdc42Hs levels (bottom panel). IB, immunoblot.