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Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides.

Xu J, Liu D, Gill G, Songyang Z - J. Cell Biol. (2001)

Bottom Line: Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo.These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides.Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

View Article: PubMed Central - PubMed

Affiliation: Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstream targets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, including cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, including its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and regulated remains to be elucidated. Unlike Akt, cytokine-independent survival kinase (CISK)/SGK3 contains a Phox homology (PX) domain. PX domains have been implicated in several cellular events involving membrane trafficking. However, their precise function remains unknown. We demonstrate here that the PX domain of CISK interacts with phosphatidylinositol (PtdIns)(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2. The CISK PX domain is required for targeting CISK to the endosomal compartment. Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo. These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides. Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

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The PX domain can regulate CISK activity. (Top) The activities of wild-type or mutant CISK were compared using luciferase assays. 293T cells were transiently transfected with a mock vector, FKHRL1 (50ng) alone, or FKHRL1 (50 ng) with either wild-type CISK (0.1 or 0.5 μg), CISK-ΔPX (0.1 or 0.5 μg) or CISK-R90A (0.1 or 0.5 μg). Error bars indicate standard errors. (Bottom) The expression of various CISK constructs was examined by Western blotting with an anti-HA antibody.
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fig4: The PX domain can regulate CISK activity. (Top) The activities of wild-type or mutant CISK were compared using luciferase assays. 293T cells were transiently transfected with a mock vector, FKHRL1 (50ng) alone, or FKHRL1 (50 ng) with either wild-type CISK (0.1 or 0.5 μg), CISK-ΔPX (0.1 or 0.5 μg) or CISK-R90A (0.1 or 0.5 μg). Error bars indicate standard errors. (Bottom) The expression of various CISK constructs was examined by Western blotting with an anti-HA antibody.

Mentions: We have demonstrated that the PX domain is required for phospholipid binding and endosomal localization of CISK. Next, we investigated if the PX domain–lipid interactions were also important for regulating the kinase activity of CISK. We have shown previously that one of the potential downstream targets of CISK is FKHRL1 (Liu et al., 2000). Phosphorylation of FKHRL1 by Akt and SGK kinases inhibits transcription activity of FKHRL1 by increasing its nuclear export, which may result in a decrease in FasL expression and hence promote cell survival (Biggs et al., 1999; Brunet et al., 1999, 2001). To test the hypothesis that the PX domain is important for the function of CISK, we examined the inhibition of FKHRL1 activity by CISK and its PX domain mutants using a luciferase reporter assay in 293T cells. As reported previously (Liu et al., 2000), CISK appeared to inhibit FKHRL1 activity in a dose-dependent manner (Fig. 4) . This inhibition was enhanced significantly with the deletion of the PX domain, indicating that the PX domain may negatively regulate CISK activity. In contrast, the R90A mutation abolished the inhibition of FKHRL1 by CISK, suggesting that phosphoinositide binding may be crucial for CISK function in vivo. These data point to a model where lipid interaction may be required for the activation of CISK or colocalization of CISK to its substrates.


Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides.

Xu J, Liu D, Gill G, Songyang Z - J. Cell Biol. (2001)

The PX domain can regulate CISK activity. (Top) The activities of wild-type or mutant CISK were compared using luciferase assays. 293T cells were transiently transfected with a mock vector, FKHRL1 (50ng) alone, or FKHRL1 (50 ng) with either wild-type CISK (0.1 or 0.5 μg), CISK-ΔPX (0.1 or 0.5 μg) or CISK-R90A (0.1 or 0.5 μg). Error bars indicate standard errors. (Bottom) The expression of various CISK constructs was examined by Western blotting with an anti-HA antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196448&req=5

fig4: The PX domain can regulate CISK activity. (Top) The activities of wild-type or mutant CISK were compared using luciferase assays. 293T cells were transiently transfected with a mock vector, FKHRL1 (50ng) alone, or FKHRL1 (50 ng) with either wild-type CISK (0.1 or 0.5 μg), CISK-ΔPX (0.1 or 0.5 μg) or CISK-R90A (0.1 or 0.5 μg). Error bars indicate standard errors. (Bottom) The expression of various CISK constructs was examined by Western blotting with an anti-HA antibody.
Mentions: We have demonstrated that the PX domain is required for phospholipid binding and endosomal localization of CISK. Next, we investigated if the PX domain–lipid interactions were also important for regulating the kinase activity of CISK. We have shown previously that one of the potential downstream targets of CISK is FKHRL1 (Liu et al., 2000). Phosphorylation of FKHRL1 by Akt and SGK kinases inhibits transcription activity of FKHRL1 by increasing its nuclear export, which may result in a decrease in FasL expression and hence promote cell survival (Biggs et al., 1999; Brunet et al., 1999, 2001). To test the hypothesis that the PX domain is important for the function of CISK, we examined the inhibition of FKHRL1 activity by CISK and its PX domain mutants using a luciferase reporter assay in 293T cells. As reported previously (Liu et al., 2000), CISK appeared to inhibit FKHRL1 activity in a dose-dependent manner (Fig. 4) . This inhibition was enhanced significantly with the deletion of the PX domain, indicating that the PX domain may negatively regulate CISK activity. In contrast, the R90A mutation abolished the inhibition of FKHRL1 by CISK, suggesting that phosphoinositide binding may be crucial for CISK function in vivo. These data point to a model where lipid interaction may be required for the activation of CISK or colocalization of CISK to its substrates.

Bottom Line: Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo.These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides.Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

View Article: PubMed Central - PubMed

Affiliation: Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstream targets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, including cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, including its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and regulated remains to be elucidated. Unlike Akt, cytokine-independent survival kinase (CISK)/SGK3 contains a Phox homology (PX) domain. PX domains have been implicated in several cellular events involving membrane trafficking. However, their precise function remains unknown. We demonstrate here that the PX domain of CISK interacts with phosphatidylinositol (PtdIns)(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2. The CISK PX domain is required for targeting CISK to the endosomal compartment. Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo. These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides. Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

Show MeSH