Report |
Address correspondence to Zhou Songyang, Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. Tel.: (713) 798-5220. Fax: (713) 796-9438. E-mail: songyang{at}bcm.tmc.edu
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Abstract |
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Key Words: phosphoinositides; PX domain; CISK/SGK3; Akt; endosome
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Introduction |
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PKB/Akt, the cellular counterpart of v-Akt, has been implicated in the regulation of cell survival in mammalian cells (Chan et al., 1999; Datta et al., 1999). Akt contains a PH domain NH2-terminal to its Ser/Thr kinase domain. The Akt PH domain has been shown to preferentially bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (James et al., 1996; Franke et al., 1997). As a result of this phospholipid binding, the conformation of Akt may be altered for subsequent effective phosphorylation by PDK1. Furthermore, Akt is recruited to the membrane, where it is colocalized with and activated by PDK1 (Chan et al., 1999). In comparison, little is known about how SGK family kinases are targeted to PDK1.
We have recently cloned an SGK family kinase cytokine-independent survival kinase (CISK)/SGK3 that shares significant homology with Akt in the kinase domain (Liu et al., 2000). Several lines of evidence suggest that CISK may be regulated in a similar fashion as Akt: (a) the two regulatory sites (TFCG and FXXFSY) which are modulated by PDK1 in Akt and SGK are also conserved in CISK (Kobayashi and Cohen, 1999; Park et al., 1999; Liu et al., 2000). (b) Similar to Akt, CISK is a survival kinase that can also protect IL-3dependent cells from apoptosis induced by IL-3 withdrawal (Songyang et al., 1997; Liu et al., 2000). (c) CISK functions downstream of the PI-3 kinase cascade and phosphorylates several Akt substrates, including the forkhead family of transcription factor FKHRL1 (Brunet et al., 1999; Kops et al., 1999; Liu et al., 2000). These observations indicate that CISK may function in parallel to Akt. However, the subcellular localization of CISK is different from that of Akt. In contrast to Akt, CISK contains a Phox homology (PX) domain NH2-terminal to its kinase domain. Although PX domains have been shown to mediate homotypic interactions (Haft et al., 1998), whether they bind phosphoinositides is not known. Interestingly, PX domains have been found in many proteins that mediate membrane functions (e.g., sorting nexins [SNX] in vesicular trafficking, and Vps5p and Vps17p in vacuolar protein sorting) (Ekena and Stevens, 1995; Ponting, 1996; Horazdovsky et al., 1997; Nothwehr and Hindes, 1997; Haft et al., 1998). In the case of CISK, the PX domain appears to be important for targeting CISK to vesicle-like structures (Liu et al., 2000).
The similarities between CISK and Akt and the subcellular localization of CISK led us to propose that the CISK PX domain may also bind phospholipids. In a manner similar to the Akt PH domain, the lipid binding ability of the CISK PX domain may be required for correctly targeting CISK to its subcellular compartment and for regulating CISK activity. To this end, we investigated the lipid binding ability of CISK PX domain and demonstrated that it could indeed bind phosphoinositides. Furthermore, this binding was important for both CISK subcellular localization and activity. These data strongly suggest that CISK has evolved to utilize a different domain to accomplish similar regulation compared with Akt.
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Results and discussion |
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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.
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Our data suggest that CISK and Akt have used different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling. Although sharing some similarities, CISK may be regulated and function distinctly from Akt. Studies of Akt have shown that Akt translocates to the plasma membrane, where it is activated upon PI-3 kinase activation and then migrates into the nucleus (Andjelkovic et al., 1997). In comparison, in growing cells CISK is mainly present in the endosomal compartments with weak nuclear staining. Both disassociation from endosomes and nuclear import may be necessary for CISK to regulate FKHRL1. Deletion of the PX domain may enhance nuclear import, which may explain why HA-CISK-PX is more active in inhibiting FKHRL1. Furthermore, the Akt PH domain and the CISK PX domain appear to have different preferences for phosphoinositides. Such differences may determine both their subcellular localization and the potential substrates that they act upon in vivo.
Different from Akt, it is possible that CISK may regulate vesicle trafficking and thereby modulate the number of activated receptors in the cell. Activated EGFRs, upon binding with EGF, are internalized and targeted for vacuolar degradation. Such membrane protein sorting is thought to occur within MVBs which form through invagination of the endosomal membrane (Futter et al., 1996; Odorizzi et al., 2000). Our observation of the comigration of EGF with CISK-containing vesicles suggests that CISK may be similar to SNX1 in the degradation process of activated receptors (Kurten et al., 1996). This hypothesis is consistent with our finding that the CISK PX domain preferentially binds to PtdIns(3,5)P2 and PtdIns(3,4,5)P3. In yeast and mammalian cells, the Fab1p kinase and its mammalian homologue PIKfyve function as PtdIns(3)P-5 kinases to generate PtdIns(3,5)P2, and have both been implicated in membrane trafficking (Yamamoto et al., 1995; Cooke et al., 1998; Gary et al., 1998; Ikonomov et al., 2001). In addition, in yeast, PtdIns(3,5)P2 appears to be required for controlling MVB sorting to the vacuole/lysosome (Odorizzi et al., 1998). The direct downstream mediators of PtdIns(3,5)P2 remain unknown. Given that CISK binds PtdIns(3,5)P2 and localizes to the relevant compartment, CISK might represent one of the targets of Fab1p and its homologues that are involved in endosomeMVB fusion with lysosomes.
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Materials and methods |
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GST fusion proteins
To generate GST PX domain fusion proteins, sequences encoding the CISK PX domain (amino acid 1136) or CISK PX domain point mutant (R90A) (generated using the Quikexchange mutagenesis kit; Stratagene) were cloned into the pGEX4T-1 vector (Amersham Pharmacia Biotech), and used to transform Escherichia coli. Expression of fusion proteins was induced by the addition of 0.2 mM IPTG. GST fusion proteins were purified from bacterial lysates using glutathione-agarose beads (Molecular Probes).
Protein/lipid overlay assays
The protein/lipid overlay assays were performed as described (Dowler et al., 1999). PIP-StripTM and PIP-ArrayTM were purchased from Echelon, Inc. For phospholipid binding, the strips or arrays were incubated overnight at 4°C with GSTPX domain fusion proteins (1 µg/ml) in TBST (10 mM Tris, 150 mM Nacl, and 0.1% Tween-20) with 3% fatty acidfree BSA (Sigma-Aldrich). The membranes were then washed in TBST with 3% fatty acidfree BSA and incubated with a monoclonal anti-GST antibody (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The membranes were then washed in TBST, incubated with a HRP-conjugated antimouse antibody (Bio-Rad Laboratories), and visualized via ECL (Amersham Pharmacia Biotech).
Immunostaining
COS-7 and 3T3 fibroblast cells grown on coverslips were fixed in 2% paraformaldehyde in 0.1 M phosphate buffer for 30 min, excess fixative was neutralized using 0.15 M glycine/PBS. COS-7 cells were blocked using 2.5% fetal calf serum/PBS for 15 min. 3T3 cells were permeabilized with 0.5% Triton X-100 in PBS and blocked with 5% normal goat serum. Primary antibodies were applied in 1% BSA (Sigma-Aldrich) or goat serum in PBS for 1 h at room temperature, followed by a corresponding Alexa 488, Alexa 594conjugated (Molecular Probes), or FITC-conjugated (Sigma-Aldrich) secondary antibody diluted 1:250 in 1% BSA/PBS for 30 min at room temperature. Omission of primary antibodies was used as negative control. The cells were also incubated with DAPI for 3 min at 25°C to stain their nuclei. For experiments with EGF, Texas redconjugated EGF (Molecular Probes) was added for initiation of internalization. The cells were then incubated at 37°C for various periods of time. The coverslips were washed with PBS, mounted onto glass microscope slides and viewed using a ZEISS Axioskop fluorescence microscope equipped with a Interline Transfer CCD Camera (COHU, Inc.). For experiments using wortmannin, the cells were first treated with 100 nM wortmannin for 0, 15, 30, or 40 min before fixation. Cell lysates were also Western blotted to confirm protein expression.
The antibodies used were: rabbit polyclonal anti-HA (1:200; Santa Cruz Biotechnology, Inc.); mouse monoclonal anti-HA (1:1,000; Babco); mouse monoclonal anti-EEA1 (1:100; Transduction Labs); and mouse monoclonal anti-LAMP (1:200; Development Studies Hybridoma Bank).
Luciferase assays
Luciferase assays were carried out as described (Liu et al., 2000). 293T cells were transiently transfected with various constructs at different concentrations. The total amount of transfected DNA was normalized using pcDNA3. The reporter construct (50 ng/transfection) contains two copies of insulin-responsive sequence from IGFBP-1 (Guo et al., 1999) in the luciferase construct pGL2. ß-gal (50 ng/transfection) was used as control to normalize transfection efficiency. At 30 h posttransfection, the cells were harvested to assay for luciferase activity as described in the manufacturer's manual (Promega). Cell lysates were also Western blotted using a monoclonal anti-HA antibody (Sigma-Aldrich) to confirm protein expression.
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Footnotes |
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* Abbreviations used in this paper: CISK, cytokine-independent survival kinase; EEA1, early endosome autoantigen 1; EGFR, EGF receptor; GST, glutathione S-transferase; HA, hemagglutinin; LAMP, lysosomal membrane glycoprotein; MVB, multivesicular body; PDK1, 3'-phophoinositidedependent kinase 1; PtdIns, phosphatidylinositol; PX, Phox homology; SGK, serum and glucocorticoidregulated kinase; SNX, sorting nexin.
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Acknowledgments |
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This work was supported by the Welch foundation (Z. Songyang) and by the National Institutes of Health grants CA84208 (Z. Songyang) and CA58689 (G. Gill). D. Liu is an American Cancer Society Postdoctoral Fellow.
Note added in proof. While this manuscript was in review, the following papers reported the interactions between PX domains and phosphoinositides: Cheever, M.L., T.K. Sato, T. de Beer, T.G. Kutateladze, S.D. Emr, and M. Overduin. Nat. Cell Biol. 3:613618; Kanai, F., H. Liu, S.J. Field, H. Akbary, T. Matsuo, G.E. Brown, L.C. Cantley, and M.B. Yaff. Nat. Cell Biol. 3:675678; Xu, Y., H. Hortsman, L. Seet, S. Heng Wong, and W. Hong. Nat. Cell Biol. 3:65866; Ellson, C.D., S. Gobert-Gosse, K.E. Anderson, K. Davidson, H. Erdjument-Bromage, P. Tempst, J.W. Thuring, M.A. Cooper, Z.Y. Lim, A.B. Holmes, et al. Nat. Cell Biol. 3:679682.
Submitted: 18 May 2001
Revised: 29 June 2001
Accepted: 9 July 2001
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