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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 CISK PX domain can specifically bind phosphoinositides. (A) The ability of the CISK PX domain to bind lipids was tested by a protein/lipid overlay assay using GST-CISK PX domain fusion proteins and nitrocellulose-immobilized phospholipid strips (100 pmole/spot). PtdIns, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine. (B) Sequence alignment of the PX domains from CISK, SNX16, SNX1, Mvp1p, Vps5, PI-3 kinase, and p40phox. Asterisk indicates conserved Arg residues. (C) The lipid binding abilities of the wild-type (left) and mutant (R90A; middle) CISK PX domain were examined using nitrocelluose-immobilized phosphoinositide arrays (100–1.6 pmole/spot with twofold dilution). The triangles indicate decreasing concentration. The expression of the GST fusion proteins is shown on the right.
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fig2: The CISK PX domain can specifically bind phosphoinositides. (A) The ability of the CISK PX domain to bind lipids was tested by a protein/lipid overlay assay using GST-CISK PX domain fusion proteins and nitrocellulose-immobilized phospholipid strips (100 pmole/spot). PtdIns, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine. (B) Sequence alignment of the PX domains from CISK, SNX16, SNX1, Mvp1p, Vps5, PI-3 kinase, and p40phox. Asterisk indicates conserved Arg residues. (C) The lipid binding abilities of the wild-type (left) and mutant (R90A; middle) CISK PX domain were examined using nitrocelluose-immobilized phosphoinositide arrays (100–1.6 pmole/spot with twofold dilution). The triangles indicate decreasing concentration. The expression of the GST fusion proteins is shown on the right.

Mentions: To test this possibility, we investigated the binding of CISK PX domain to a collection of phospholipids. Consistent with our hypothesis, CISK could indeed bind phosphoinositides, but not phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, PtdIns, or phosphatidylcholine (Fig. 2 A). The sequence alignment of multiple PX domains suggests that residue R90 is most conserved among PX domains (Fig. 2 B) and may be necessary for phospholipid binding. To further determine the relative preference of the CISK PX domain to various phosphoinositides, wild-type and mutant (R90A) glutathione S-transferase (GST)-PX fusion proteins were used to probe arrays containing different concentrations of phospholipids. As shown in Fig. 2 C, the CISK PX domain could specifically interact with PtdIns(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2, whereas the R90A mutation greatly diminished the binding. These results demonstrate that the PX domain of CISK could directly interact with specific phosphoinositides. The observation that the CISK PX domain selectively bound PtdIns(3,5)P2 but not PtdIns(3,4)P2 might explain the differential subcellular localization of CISK and Akt.


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 CISK PX domain can specifically bind phosphoinositides. (A) The ability of the CISK PX domain to bind lipids was tested by a protein/lipid overlay assay using GST-CISK PX domain fusion proteins and nitrocellulose-immobilized phospholipid strips (100 pmole/spot). PtdIns, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine. (B) Sequence alignment of the PX domains from CISK, SNX16, SNX1, Mvp1p, Vps5, PI-3 kinase, and p40phox. Asterisk indicates conserved Arg residues. (C) The lipid binding abilities of the wild-type (left) and mutant (R90A; middle) CISK PX domain were examined using nitrocelluose-immobilized phosphoinositide arrays (100–1.6 pmole/spot with twofold dilution). The triangles indicate decreasing concentration. The expression of the GST fusion proteins is shown on the right.
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Related In: Results  -  Collection

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

fig2: The CISK PX domain can specifically bind phosphoinositides. (A) The ability of the CISK PX domain to bind lipids was tested by a protein/lipid overlay assay using GST-CISK PX domain fusion proteins and nitrocellulose-immobilized phospholipid strips (100 pmole/spot). PtdIns, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine. (B) Sequence alignment of the PX domains from CISK, SNX16, SNX1, Mvp1p, Vps5, PI-3 kinase, and p40phox. Asterisk indicates conserved Arg residues. (C) The lipid binding abilities of the wild-type (left) and mutant (R90A; middle) CISK PX domain were examined using nitrocelluose-immobilized phosphoinositide arrays (100–1.6 pmole/spot with twofold dilution). The triangles indicate decreasing concentration. The expression of the GST fusion proteins is shown on the right.
Mentions: To test this possibility, we investigated the binding of CISK PX domain to a collection of phospholipids. Consistent with our hypothesis, CISK could indeed bind phosphoinositides, but not phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, PtdIns, or phosphatidylcholine (Fig. 2 A). The sequence alignment of multiple PX domains suggests that residue R90 is most conserved among PX domains (Fig. 2 B) and may be necessary for phospholipid binding. To further determine the relative preference of the CISK PX domain to various phosphoinositides, wild-type and mutant (R90A) glutathione S-transferase (GST)-PX fusion proteins were used to probe arrays containing different concentrations of phospholipids. As shown in Fig. 2 C, the CISK PX domain could specifically interact with PtdIns(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2, whereas the R90A mutation greatly diminished the binding. These results demonstrate that the PX domain of CISK could directly interact with specific phosphoinositides. The observation that the CISK PX domain selectively bound PtdIns(3,5)P2 but not PtdIns(3,4)P2 might explain the differential subcellular localization of CISK and Akt.

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
Related in: MedlinePlus