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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 lipid-binding PX domain is important for CISK localization. (A) The vesicular subcellular localization of CISK is disrupted after wortmannin treatment. 3T3 cells expressing HA-tagged wild-type CISK were treated with wortmannin (100 nM) for 0, 15, 30, or 40 min before fixation for immunostaining analysis. (B) The localization of HA-tagged wild-type CISK, CISK-ΔPX, and CISK-R90A mutants in 3T3 cells was compared.
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fig3: The lipid-binding PX domain is important for CISK localization. (A) The vesicular subcellular localization of CISK is disrupted after wortmannin treatment. 3T3 cells expressing HA-tagged wild-type CISK were treated with wortmannin (100 nM) for 0, 15, 30, or 40 min before fixation for immunostaining analysis. (B) The localization of HA-tagged wild-type CISK, CISK-ΔPX, and CISK-R90A mutants in 3T3 cells was compared.

Mentions: Because the CISK PX domain could bind PtdIns(3,5)P2 and PtdIns(3,4,5)P3, we speculated that such binding might be essential for the subcellular localization of CISK. CISK colocalizes with EEA1, a protein shown to be targeted to the endosomes through interaction with PtdIns(3)P via its FYVE domain (Patki et al., 1997; Burd and Emr, 1998; Simonsen et al., 1998). Since the production of D3-phosphoinositides is dependent on PI-3 kinase, we first examined CISK localization upon treatment with the PI-3 kinase inhibitor wortmannin. As shown in Fig. 3 A, the endosomal distribution of CISK was disrupted over time upon wortmannin treatment. At 40 min after treatment, vesicular distribution of CISK was no longer detectable. Therefore, the localization of CISK to the endosomes likely requires D3-phosphoinositides, consistent with our result that the CISK PX domain preferentially binds D3-phosphoinositides PtdIns(3,5)P2 and PtdIns(3,4,5)P3. In addition, deletion of the PX domain abolished the vesicular localization of CISK (Fig. 3 B). Furthermore, the CISK mutant (R90A) that failed to bind phospholipids was not targeted to the endosomes (Fig. 3 B). In contrast, full-length kinase-dead CISK with a mutation in the ATP binding site still localized to the vesicles (data not shown), suggesting that the kinase activity is not required for CISK targeting. These data combined indicate strongly that the PX domain of CISK is crucial in regulating the endosomal localization of CISK, and this regulation is most likely phosphoinositide-dependent.


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 lipid-binding PX domain is important for CISK localization. (A) The vesicular subcellular localization of CISK is disrupted after wortmannin treatment. 3T3 cells expressing HA-tagged wild-type CISK were treated with wortmannin (100 nM) for 0, 15, 30, or 40 min before fixation for immunostaining analysis. (B) The localization of HA-tagged wild-type CISK, CISK-ΔPX, and CISK-R90A mutants in 3T3 cells was compared.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196448&req=5

fig3: The lipid-binding PX domain is important for CISK localization. (A) The vesicular subcellular localization of CISK is disrupted after wortmannin treatment. 3T3 cells expressing HA-tagged wild-type CISK were treated with wortmannin (100 nM) for 0, 15, 30, or 40 min before fixation for immunostaining analysis. (B) The localization of HA-tagged wild-type CISK, CISK-ΔPX, and CISK-R90A mutants in 3T3 cells was compared.
Mentions: Because the CISK PX domain could bind PtdIns(3,5)P2 and PtdIns(3,4,5)P3, we speculated that such binding might be essential for the subcellular localization of CISK. CISK colocalizes with EEA1, a protein shown to be targeted to the endosomes through interaction with PtdIns(3)P via its FYVE domain (Patki et al., 1997; Burd and Emr, 1998; Simonsen et al., 1998). Since the production of D3-phosphoinositides is dependent on PI-3 kinase, we first examined CISK localization upon treatment with the PI-3 kinase inhibitor wortmannin. As shown in Fig. 3 A, the endosomal distribution of CISK was disrupted over time upon wortmannin treatment. At 40 min after treatment, vesicular distribution of CISK was no longer detectable. Therefore, the localization of CISK to the endosomes likely requires D3-phosphoinositides, consistent with our result that the CISK PX domain preferentially binds D3-phosphoinositides PtdIns(3,5)P2 and PtdIns(3,4,5)P3. In addition, deletion of the PX domain abolished the vesicular localization of CISK (Fig. 3 B). Furthermore, the CISK mutant (R90A) that failed to bind phospholipids was not targeted to the endosomes (Fig. 3 B). In contrast, full-length kinase-dead CISK with a mutation in the ATP binding site still localized to the vesicles (data not shown), suggesting that the kinase activity is not required for CISK targeting. These data combined indicate strongly that the PX domain of CISK is crucial in regulating the endosomal localization of CISK, and this regulation is most likely phosphoinositide-dependent.

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