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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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CISK is localized to endosomal compartments. COS-7 cells transiently transfected with HA-epitope–tagged CISK were examined for the localization of (A) CISK (green) and endogenous EEA1 (red), and (B) CISK (green) and endogenous LAMP (red). Merged images are shown on the right. (C) Texas red–conjugated EGF was added to COS-7 cells expressing HA-CISK. The subcellular localization of CISK (green) and the migration of EGF (red; arrowheads) was examined at 0, 5, and 15 min after EGF addition at 37°C. The migration of EGF with CISK-containing vesicles is indicated by arrows.
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fig1: CISK is localized to endosomal compartments. COS-7 cells transiently transfected with HA-epitope–tagged CISK were examined for the localization of (A) CISK (green) and endogenous EEA1 (red), and (B) CISK (green) and endogenous LAMP (red). Merged images are shown on the right. (C) Texas red–conjugated EGF was added to COS-7 cells expressing HA-CISK. The subcellular localization of CISK (green) and the migration of EGF (red; arrowheads) was examined at 0, 5, and 15 min after EGF addition at 37°C. The migration of EGF with CISK-containing vesicles is indicated by arrows.

Mentions: CISK is a PX domain–containing Ser/Thr kinase that belongs to the SGK family of kinases (Liu et al., 2000). The exact function of PX domains is not yet known. Most PX domains are found in proteins involved in trafficking, such as SNXs (Ponting, 1996). Consistent with this observation, we have found CISK to localize in vesicle-like structures (Liu et al., 2000). Such localization is distinct from that of Akt (Andjelkovic et al., 1997) and suggests that the PX domain may play an important role in targeting CISK. Further examination of these vesicles revealed that CISK was targeted to the endosomal compartments. As shown in Fig. 1 , CISK (green) was found to colocalize with the endosome marker human EEA1 (red, Fig. 1 A), but not the lysosomal marker lysosomal membrane glycoprotein (LAMP) (red, Fig. 1 B). Colocalization of hemagglutinin (HA)-CISK and EEA1 was also verified by deconvolution immunofluorescence microscopy. Because EGF-bound EGF receptors (EGFRs) interact with SNX1 and are sorted by multivesicular bodies (MVBs) formed by endosomal membranes (Haigler et al., 1979; Futter et al., 1996; Kurten et al., 1996), we examined if EGF would colocalize with CISK-containing vesicles. At t = 0, EGF was bound to EGFR at the cell surface (arrowheads, Fig. 1 C). At 37°C, EGF is rapidly internalized. Interestingly, as early as 5 min after EGF addition, vesicles that contained both EGF and CISK could be detected. After 15 min, most of the EGF appeared to transit through the CISK-containing vesicles (arrows). These observations suggest that EGF enters cells via receptor-mediated endocytosis and then enters the CISK-containing endosome compartment.


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)

CISK is localized to endosomal compartments. COS-7 cells transiently transfected with HA-epitope–tagged CISK were examined for the localization of (A) CISK (green) and endogenous EEA1 (red), and (B) CISK (green) and endogenous LAMP (red). Merged images are shown on the right. (C) Texas red–conjugated EGF was added to COS-7 cells expressing HA-CISK. The subcellular localization of CISK (green) and the migration of EGF (red; arrowheads) was examined at 0, 5, and 15 min after EGF addition at 37°C. The migration of EGF with CISK-containing vesicles is indicated by arrows.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: CISK is localized to endosomal compartments. COS-7 cells transiently transfected with HA-epitope–tagged CISK were examined for the localization of (A) CISK (green) and endogenous EEA1 (red), and (B) CISK (green) and endogenous LAMP (red). Merged images are shown on the right. (C) Texas red–conjugated EGF was added to COS-7 cells expressing HA-CISK. The subcellular localization of CISK (green) and the migration of EGF (red; arrowheads) was examined at 0, 5, and 15 min after EGF addition at 37°C. The migration of EGF with CISK-containing vesicles is indicated by arrows.
Mentions: CISK is a PX domain–containing Ser/Thr kinase that belongs to the SGK family of kinases (Liu et al., 2000). The exact function of PX domains is not yet known. Most PX domains are found in proteins involved in trafficking, such as SNXs (Ponting, 1996). Consistent with this observation, we have found CISK to localize in vesicle-like structures (Liu et al., 2000). Such localization is distinct from that of Akt (Andjelkovic et al., 1997) and suggests that the PX domain may play an important role in targeting CISK. Further examination of these vesicles revealed that CISK was targeted to the endosomal compartments. As shown in Fig. 1 , CISK (green) was found to colocalize with the endosome marker human EEA1 (red, Fig. 1 A), but not the lysosomal marker lysosomal membrane glycoprotein (LAMP) (red, Fig. 1 B). Colocalization of hemagglutinin (HA)-CISK and EEA1 was also verified by deconvolution immunofluorescence microscopy. Because EGF-bound EGF receptors (EGFRs) interact with SNX1 and are sorted by multivesicular bodies (MVBs) formed by endosomal membranes (Haigler et al., 1979; Futter et al., 1996; Kurten et al., 1996), we examined if EGF would colocalize with CISK-containing vesicles. At t = 0, EGF was bound to EGFR at the cell surface (arrowheads, Fig. 1 C). At 37°C, EGF is rapidly internalized. Interestingly, as early as 5 min after EGF addition, vesicles that contained both EGF and CISK could be detected. After 15 min, most of the EGF appeared to transit through the CISK-containing vesicles (arrows). These observations suggest that EGF enters cells via receptor-mediated endocytosis and then enters the CISK-containing endosome compartment.

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