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. 2009 Jul 10;284(36):24512–24525. doi: 10.1074/jbc.M109.026237

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© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — 14-3-3 interacts with Akt-phosphorylated SRPK2 and inhibits its nuclear translocation. A, SRPK2 binds to 14-3-3. HEK293 cells were transfected with Myc-SRPK2 and a variety of HA-14-3-3 isoforms. 14-3-3 was immunoprecipitated using HA antibody and was detected with Myc antibody (top panel). Expression of Myc-SRPK2 and HA-14-3-3s was verified (middle and bottom panels). IP, immunoprecipitate; NS, nonspecific. B, PI 3-kinase signaling regulates the interaction between endogenous SRPK2 and 14-3-3β. HEK293 cells were pretreated with PD98059, wortmannin, LY294002 for 30 min before EGF was introduced. Endogenous SRPK2 was immunoprecipitated and analyzed using anti-14-3-3β-specific antibody. The relative signal strength is labeled underneath of each blot. Equal amounts of SRPK2 were immunoprecipitated (second panel). Confirmation of Akt and SRPK2 phosphorylation status (third and fifth panels). C, SRPK2 binding to 14-3-3 is Akt phosphorylation-dependent. Myc-SRPK2 T492A and T492D were cotransfected into 293 cells with HA-14-3-3β, -σ, and -ϵ isoforms. 14-3-3 was immunoprecipitated using HA antibody and detected with Myc antibody. 14-3-3σ bound to both SRPK2 T492A and T492D proteins.14-3-3β and -ϵ isoforms selectively bound to SRPK2/T492D but not T492A (top panel). The expression of Myc-SRPK2 and HA-14-3-3s were examined (middle and bottom panels). D, 14-3-3 K50E mutant fails to interact with SRPK2. SRPK2 construct alone or together with HA-14-3-3 wild type or K50E mutant was transfected into HEK293 cells. 14-3-3 was immunoprecipitated using HA antibody and detected using Myc antibody (top panel). The expression of Myc-SRPK2, HA-14-3-3 wild type and mutant was examined (middle and bottom panels). E, Akt phosphorylation regulates SRPK2 subcellular location. Various HA-Akt constructs (WT, CA (constitutively active), KD) were cotransfected with SRPK2 wild type into HEK293 cells and monitored by immunofluorescent staining. F, effects of 14-3-3 on intracellular localization of SRPK2. Myc-SRPK2 wild type, T492A, and T492D were cotransfected with HA-14-3-3β into 293 cells, and indirect immunofluorescent staining was performed. Cotransfection with HA-14-3-3 inhibited SRPK2 T492D from translocating into the nucleus. The experiments described in E and F have been done three times. In total, 100 cells in E and 150 cells in F, were counted.