Jee-Yin Ahn

Akt/PKB is a crucial regulator of diverse cellular processes and contributes to cancer progression. Activation of Akt is essentially dependent on phosphatidylinositol (PI) 3-kinase signaling. Here, we describe a novel mediator of Akt that is independent of PI 3-kinase. This mediator, PIKE-A, is a PIKE isoform and contains GTPase, pleckstrin homology, ArfGAP, and ankyrin repeats domains. PIKE-A directly binds to activated Akt but not PI 3-kinase in a guanine nucleotide-dependent way and stimulates the kinase activity of Akt. Overexpression of PIKE-A enhances Akt activity and promotes cancer cell invasion, whereas dominant-negative PIKE-A and PIKE-A knockdown markedly inhibit these processes. Our results demonstrate that PIKE-A is a physiologic regulator of Akt and an oncogenic effector of cell invasion. Akt/PKB is a crucial regulator of diverse cellular processes and contributes to cancer progression. Activation of Akt is essentially dependent on phosphatidylinositol (PI) 3-kinase signaling. Here, we describe a novel mediator of Akt that is independent of PI 3-kinase. This mediator, PIKE-A, is a PIKE isoform and contains GTPase, pleckstrin homology, ArfGAP, and ankyrin repeats domains. PIKE-A directly binds to activated Akt but not PI 3-kinase in a guanine nucleotide-dependent way and stimulates the kinase activity of Akt. Overexpression of PIKE-A enhances Akt activity and promotes cancer cell invasion, whereas dominant-negative PIKE-A and PIKE-A knockdown markedly inhibit these processes. Our results demonstrate that PIKE-A is a physiologic regulator of Akt and an oncogenic effector of cell invasion. Phosphatidylinositol 3-kinase enhancer (PIKE) 1The abbreviations used are: PI, phosphatidylinositol; PIKE, phosphatidylinositol 3-kinase enhancer; PH, pleckstrin homology; mGluR, metabotropic glutamate receptors; HA, hemagglutinin; GST, glutathione s-transferase; FISH, fluorescent in situ hybridization; WT, wildtype; DN, dominant-negative; Mes, 2-(N-morpholino)ethanesulfonic acid; EGF, epidermal growth factor; IGF-1, insulin-linked growth factor 1; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GDPβS, guanyl-5′-yl thiophosphate; ATPγS, adenosine 5′-O-(thiotriphosphate); JNK, c-Jun N-terminal kinase; Cdk, cyclin-dependent kinasek Erk, extracellular signal-regulated kinase. 1The abbreviations used are: PI, phosphatidylinositol; PIKE, phosphatidylinositol 3-kinase enhancer; PH, pleckstrin homology; mGluR, metabotropic glutamate receptors; HA, hemagglutinin; GST, glutathione s-transferase; FISH, fluorescent in situ hybridization; WT, wildtype; DN, dominant-negative; Mes, 2-(N-morpholino)ethanesulfonic acid; EGF, epidermal growth factor; IGF-1, insulin-linked growth factor 1; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GDPβS, guanyl-5′-yl thiophosphate; ATPγS, adenosine 5′-O-(thiotriphosphate); JNK, c-Jun N-terminal kinase; Cdk, cyclin-dependent kinasek Erk, extracellular signal-regulated kinase. is a recently discovered protein, which possesses GTPase activity and stimulates the lipid kinase activity of nuclear PI 3-kinase (1Ye K. Hurt K.J. Wu F.Y. Fang M. Luo H.R. Hong J.J. Blackshaw S. Ferris C.D. Snyder S.H. Cell. 2000; 103: 919-930Abstract Full Text Full Text PDF PubMed Google Scholar). In PC12 cells treated with nerve growth factor, activation of nuclear PI 3-kinase is mediated by PIKE. PIKE was originally identified in a yeast two-hybrid screening searching for binding partners of the C-terminal domain of protein 4.1N. Nerve growth factor elicits 4.1N to translocate to the nucleus where it binds PIKE and regulates the effects of PIKE on PI 3-kinase. Phospholipase C-γ1 binds PIKE on its third proline-rich domain and acts as a physiological guanine-nucleotide-exchange factor for PIKE. This action is not dependent on the lipase activity of the phospholipase C-γ1 but appears instead to involve its SH3 domain (2Smith M.R. Liu Y.L. Matthews N.T. Rhee S.G. Sung W.K. Kung H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6554-6558Crossref PubMed Scopus (105) Google Scholar, 3Ye K. Aghdasi B. Luo H.R. Moriarity J.L. Wu F.Y. Hong J.J. Hurt K.J. Bae S.S. Suh P.G. Snyder S.H. Nature. 2002; 415: 541-544Crossref PubMed Scopus (146) Google Scholar, 4Huang P.S. Davis L. Huber H. Goodhart P.J. Wegrzyn R.E. Oliff A. Heimbrook D.C. FEBS Lett. 1995; 358: 287-292Crossref PubMed Scopus (70) Google Scholar). Recently, we identified an alternatively spliced form of PIKE, PIKE-L (bankit475414 (AY128689, human)), which is several hundred amino acids longer than the original form of PIKE, which we now designate PIKE-S. In addition to the GTPase and PH domains shared by PIKE-S and PIKE-L, PIKE-L contains an ArfGAP domain and two ankyrin repeats. PIKE-L binds to Homer, an adaptor protein known to link metabotropic glutamate receptors (mGluR I) to multiple intracellular targets including the inositol 1,4,5-trisphosphate receptor (5Tu J.C. Xiao B. Yuan J.P. Lanahan A.A. Leoffert K. Li M. Linden D.J. Worley P.F. Neuron. 1998; 21: 717-726Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 6Xiao B. Tu J.C. Petralia R.S. Yuan J.P. Doan A. Breder C.D. Ruggiero A. Lanahan A.A. Wenthold R.J. Worley P.F. Neuron. 1998; 21: 707-716Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, 7Tu J.C. Xiao B. Naisbitt S. Yuan J.P. Petralia R.S. Brakeman P. Doan A. Aakalu V.K. Lanahan A.A. Sheng M. Worley P.F. Neuron. 1999; 23: 583-592Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar, 8Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar). We have shown that activation of mGluR I enhances formation of an mGluR I-Homer-PIKE-L complex leading to activation of PI 3-kinase activity and prevention of neuronal apoptosis (9Rong R. Ahn J.Y. Huang H. Nagata E. Kalman D. Kapp J.A. Tu J. Worley P.F. Snyder S.H. Ye K. Nat. Neurosci. 2003; 6: 1153-1161Crossref PubMed Scopus (246) Google Scholar). Akt or protein kinase B is a major downstream target of the PI 3-kinase pathway, and plays a key role in a wide variety of cellular functions including metabolism, cell survival, and proliferation (10Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar, 11Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 12Duronio V. Scheid M.P. Ettinger S. Cell. Signal. 1998; 10: 233-239Crossref PubMed Scopus (186) Google Scholar). Akt is fully activated by phosphorylation of threonine 308 and serine 473 upon activation of PI 3-kinase signaling. Akt is frequently constitutively active in many human cancers. Constitutively active Akt occurs because of amplification of Akt genes or as a result of mutations in components of the signaling pathway that activates Akt (13Nicholson K.M. Anderson N.G. Cell. Signal. 2002; 14: 381-395Crossref PubMed Scopus (1381) Google Scholar). Akt is a crucial player in regulating diverse tumorigenic activities such as angiogenesis and tissue invasion/metastasis (14Testa J.R. Bellacosa A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10983-10985Crossref PubMed Scopus (770) Google Scholar). In the present study we have identified a novel form of PIKE, designated PIKE-A. PIKE-A is co-amplified with CDK4 in a variety of human cancers, and specifically binds to activated Akt but not PI 3-kinase in a GTP-dependent manner, and stimulates Akt kinase activity. The PIKE-A/Akt interaction is mediated through the GTPase domain of PIKE-A and the C-terminal regulatory domain and a portion of the catalytic domain of Akt. Overexpression of wild-type PIKE-A enhances Akt activity, whereas dominant-negative PIKE-A and PIKE-A knockdown inhibit it. Overexpression of wild-type and dominant-negative PIKE-A and PIKE-A knockdown show that PIKE-A regulates human cancer cell invasion, which is dependent upon the Akt. Cells and Reagents—HEK293 cells and human glioblastoma LN-Z308, LN487, LN382, SF188, SF767, and U87MG cells were maintained in Dulbecco's modified Eagle's medium, whereas neuroblastoma NGP-127 and sarcoma CRL-2061 cells were cultured in RPMI1640, supplemented with 10% fetal bovine serum, 2 mg/ml glutamine, and 100 units of penicillin-streptomycin at 37 °C with 5% CO2 atmosphere in a humidified incubator. Mouse monoclonal anti-HA-horseradish peroxidase, anti-Myc-horseradish peroxidase, and anti-GST antibodies were from Sigma. Mouse monoclonal anti-Ser473, anti-Thr308, anti-Akt, and anti-phospho-GSK3α/β (Ser-21/9) antibodies were from Cell Signaling. Rabbit polyclonal anti-p85 and p110 antibodies were from Santa Cruz Biotechnology, Inc. Anti-PIKE-C and anti-PIKE-N antibodies were raised against GST-PIKE-L (amino acids 1095–1186) and His-PIKE-L (amino acids 268–384) recombinant proteins. Protein A/G-conjugated agarose beads were from Calbiochem. Glutathione-Sepharose 4B was supplied by Amersham Biosciences. GST-GSK3 fusion protein and Crosstide were from Cell Signaling. All the chemicals not included above were from Sigma. Fluorescent in Situ Hybridization (FISH) Staining—Experimental procedures for FISH staining on human cancer cell lines are as J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). was as a for PIKE and was used as a for and in procedures for and in binding are as K. Snyder S.H. J. Neurosci. 1999; PubMed Google Scholar). the GTP-dependent and were as J.A. Cell. Full Text PDF PubMed Scopus Google Scholar). In for Akt cells or glioblastoma cells were with and DN, the cells were and with or growth factor for and with and in mg/ml mg/ml mg/ml and were at for at Cell were for protein bovine as a of cell were with and protein beads for 2 at were with and with kinase 2 were in of kinase supplemented with and of at °C for was and in and in the was was by of GST-GSK3 fusion protein as the kinase were by and for were on 10% and with anti-phospho-GSK3α/β (Ser-21/9) staining was as D. A. Nat. Cell 1999; PubMed Scopus Google Scholar). cells were for at in a that and Mes, and 2 Cells were in and for in supplemented with Cells were in and in for The antibodies and used in study were as and monoclonal antibodies and were as and from In PI PI 3-kinase was by from cell as and with the with 2 with B 2 with The procedures were as (1Ye K. Hurt K.J. Wu F.Y. Fang M. Luo H.R. Hong J.J. Blackshaw S. Ferris C.D. Snyder S.H. Cell. 2000; 103: 919-930Abstract Full Text Full Text PDF PubMed Google Scholar). Cell cells were on tissue cells were by and with of of in 2 of cells were by Cells were and in at 2 of cell was to to of were for 2 with 2 of Dulbecco's modified Eagle's 10% fetal bovine The with cells was for at 37 CO2 The was in The of the cells was on with Dulbecco's modified Eagle's The of the was with a and Dulbecco's modified Eagle's to Cells on the of the were and with and and were the and of PIKE-A an at the were to as J. Neurosci. 2001; 21: PubMed Google Scholar). The were in an of was and the was at 37 °C for The of the by by was The was cells are as of or independent and the were with PIKE in is frequently in human and S.H. P.S. Google Scholar, J. K. P.S. 1994; Google Scholar). contains two and the CDK4 M. K. R. P.S. PubMed Scopus Google Scholar, A. A. P.S. PubMed Scopus Google Scholar, K. J. P.S. Google Scholar). that the PIKE is at to CDK4 1995; PubMed Scopus Google Scholar, B. P.S. PubMed Scopus Google Scholar). In of PIKE amplification in the human glioblastoma we have identified a PIKE form which was originally identified in the human as K. A. PubMed Scopus Google and was identified by Liu and C. Liu C. L. Liu M. Cell. 2003; 23: PubMed Scopus Google Scholar). PIKE-A, and from the PIKE Recently, we have shown that PIKE-L and are alternatively spliced of PIKE (9Rong R. Ahn J.Y. Huang H. Nagata E. Kalman D. Kapp J.A. Tu J. Worley P.F. Snyder S.H. Ye K. Nat. Neurosci. 2003; 6: 1153-1161Crossref PubMed Scopus (246) Google Scholar). that PIKE-A results from from that of PIKE-L and PIKE-A contains the GTPase, PH, ArfGAP, and two ankyrin domains present in PIKE-L but the proline-rich domain the which binds protein PI and phospholipase C-γ1 PIKE we FISH on a variety of human sarcoma and cell PIKE is in the neuroblastoma cell NGP-127 and glioblastoma cell with a of PIKE on We have results on the human glioblastoma cell CRL-2061 not and in The of with PIKE amplification is in amplification of PIKE in these cancer cell we with for the PIKE-A isoform amino acids PIKE-A is in and was as a with a from the proline-rich domain of PIKE-L and amino acids two with and in to PIKE-L and with the PIKE-L in PIKE-L is in cells in the of PIKE-S with that PIKE-A is in these cancer cells in the of PIKE-L PIKE-L but not PIKE-A is in the with anti-PIKE-N in PIKE-L and PIKE-L in cells with is in and cell and cell cell lines in a PIKE-A binds to and activates nuclear PI which is a portion of the signaling that acts through the protein kinase Akt (10Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar, 11Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 12Duronio V. Scheid M.P. Ettinger S. Cell. Signal. 1998; 10: 233-239Crossref PubMed Scopus (186) Google Scholar). PIKE-A the N-terminal domain that binds to PI 3-kinase. PIKE-A, but not PIKE-L or is in human we that PIKE-A directly with Akt in the of PI 3-kinase we a in human cancer cell with an to Akt to the of PIKE-A in two human cancer cell NGP-127 and which PIKE-A. was not in a third cell which not PIKE-A and from cells that Akt specifically with PIKE-A Akt is frequently constitutively active in many of human with monoclonal that Akt is in of these cells PIKE-A and Akt in we and the sarcoma cell and of the two in the and nucleus N-terminal to the upon and Akt were in cells treated with that Akt acts as the Akt. for Akt and PIKE-A in the cells in the and nucleus third The the and in glioblastoma cells because of the PIKE-A, from PIKE the Akt but in PIKE-A and Akt are In to the nuclear of PIKE-A occurs in the and with the that PIKE-A in and nucleus C. Liu C. L. Liu M. Cell. 2003; 23: PubMed Scopus Google Scholar). which is co-amplified with PIKE-A in a PIKE-A with Akt in a with PI 3-kinase in a GTP-dependent way (1Ye K. Hurt K.J. Wu F.Y. Fang M. Luo H.R. Hong J.J. Blackshaw S. Ferris C.D. Snyder S.H. Cell. 2000; 103: 919-930Abstract Full Text Full Text PDF PubMed Google Scholar). We PIKE-A binding to Akt is by guanine we an in binding recombinant We glutathione beads to with of cells with by to recombinant is in cells treated with the with and binding of activated and PIKE-A with binding in the of guanine In of the guanine binding is dependent on a and with of or the or leading to dominant-negative that to but to targets (1Ye K. Hurt K.J. Wu F.Y. Fang M. Luo H.R. Hong J.J. Blackshaw S. Ferris C.D. Snyder S.H. Cell. 2000; 103: 919-930Abstract Full Text Full Text PDF PubMed Google Scholar). We an dominant-negative by the and serine of PIKE-A. This designated as binds Akt in cells not treated by the growth factor in treated binding occurs in the of guanine The phosphorylation of Akt with its with recombinant and a we the with and binding of guanine or We have the phosphorylation of Akt in cells treated with or PIKE-A binds to activated that Akt phosphorylation a role in its with PIKE-A. we cells with and wild-type or with threonine 308 and serine 473 to and the cells with or that binding by PIKE-A to wild-type but not Akt is in cells treated with with cells of PIKE-A and Akt are and The GTPase of PIKE-A the portion of PIKE-A that binds we cells with of PIKE with and the cells with or for The protein was by glutathione of the that the activated Akt binds to PIKE-A in growth cells with the binding is for the dominant-negative and PIKE-A the N-terminal amino acids in is dependent on the GTPase domain of PIKE-A, as it is in the form of PIKE-A domain the C-terminal ArfGAP and two ankyrin domains with Akt or not it This that the PH domain the C-terminal its interaction with Akt. The form of PIKE-A with the N-terminal with Akt of growth factor that the N-terminal portion PIKE-A binding activity in to We have the of the in of cells of occurs in growth cells but not in cells We have the binding activity with not the GTPase domain binds we an in binding with We glutathione beads to with of cells with which was with or for The interaction the GTPase domain and Akt is not by the of or GDPβS, of Akt a we to the binding we not Akt binding This result that the GTPase domain is to Akt. The wild-type PIKE-A that binds Akt upon growth factor that interaction is physiological PIKE-A with Akt through and which portion of Akt is for its with PIKE-A, we a variety of Akt and cells with The cells were and with for The fusion were by glutathione and of the that Akt and Akt the regulatory and at the catalytic domain PIKE-A upon growth factor whereas the PH and catalytic domains by not the regulatory and a portion of the catalytic domain of Akt to the crucial binding growth factor we to interaction Akt and PIKE-A in cells not The of Akt and PIKE-A were by of Akt its with PIKE-A and the regulatory domain is for portion is upon growth factor we cells with Akt and with of the that serine 473 is on the regulatory domain but not on which it of were PIKE-A Akt but PI the of PIKE-A/Akt we Akt kinase activity in cells with PIKE-A or dominant-negative elicits a of Akt activity. of wild-type PIKE-A to a of activity. with Akt activity by than In cells not treated with growth factor, markedly Akt activity results were with not We have of Akt for in kinase and of PIKE-A The GTPase domain and C-terminal domains of PIKE-A Akt and which domain is for kinase activity, we the in kinase with GST-GSK3 as a on Akt in the of PIKE-A but not or recombinant protein stimulates Akt activity of guanine on Akt activity not of fusion were the that PIKE-A binds Akt and but PIKE-A enhances Akt activity, we the in binding with active Akt and with ATPγS, GTPγS, and Akt is with guanine PIKE-A. of Akt to the binding of Akt to PIKE-A that was than by or third with or Akt kinase activity occurs the with that by is that by with against Akt these that the GTPase domain of PIKE-A stimulates Akt kinase activity in the of with the that PIKE-A enhances Akt activity in cells of growth factor PIKE-A binds active Akt and enhances its activity through Akt active or its we intracellular a that and used for knockdown J. Neurosci. 2001; 21: PubMed Google Scholar). U87MG cells were treated with to knockdown PIKE-A, whereas was as a Akt phosphorylation is in but not cells of Akt were with markedly the of PIKE-A. a the of is not results were in cells as not demonstrate that PIKE-A plays an role in and Akt PIKE-A Akt by binding and activity of nuclear PI of the Akt activation We by of and p110 of PI 3-kinase with PIKE-S or PIKE-A. PIKE-S binds and p110 of PI 3-kinase PIKE-A to to protein, with its the N-terminal portion of PIKE-S that such binding (1Ye K. Hurt K.J. Wu F.Y. Fang M. Luo H.R. Hong J.J. Blackshaw S. Ferris C.D. Snyder S.H. Cell. 2000; 103: 919-930Abstract Full Text Full Text PDF PubMed Google Scholar). The of PI 3-kinase and PIKE-A with its to PI PIKE-A not PI 3-kinase activity. PIKE-S markedly enhances PI 3-kinase activity. a PI 3-kinase activity was markedly by its PIKE-A Cell through but not PIKE-S or is in human Akt plays an role in and including cancer and (14Testa J.R. Bellacosa A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10983-10985Crossref PubMed Scopus (770) Google Scholar, A.A. A. M. 2000; PubMed Scopus Google Scholar, K. M. J. M. S. S. 2002; Google Scholar, D. P. 2003; PubMed Scopus Google Scholar). the of interaction PIKE-A and we the of PIKE-A on of cancer cells We two human glioblastoma cell LN-Z308, in which PIKE-A is and and U87MG that not PIKE-A. of U87MG cells with an wild-type PIKE-A elicits a of cell a occurs with the because PIKE-A is In and U87MG with cell by with We have essentially the results in human cancer cell SF188, SF767, and CRL-2061 not the effects for and U87MG cells because of the in cell growth the we the cell proliferation and a growth for and U87MG cells of PIKE-A not the proliferation the not In the and U87MG cells for cell we the phosphorylation of Akt. with wild-type PIKE-A markedly of and in cell lines Akt kinase activity, by the phosphorylation of a downstream target of is by wild-type PIKE-A. we or in cells with In cells the of and are by with the of Akt activation by PIKE-A, we the phosphorylation of the phosphorylation of but not is by wild-type PIKE-A, that are by PIKE-A. PIKE-A stimulates cell through we dominant-negative Akt with dominant-negative Akt cell for and U87MG the role of PIKE-A in cell invasion, we knockdown PIKE-A J. Neurosci. 2001; 21: PubMed Google Scholar). Cell is markedly in but not cells but not PIKE-A in is not with Akt phosphorylation is in but not cells Our is that PIKE-A binds to Akt in a its kinase activity, and is a physiologic mediator of Akt on cellular invasion. We the of PIKE-A with because of the of PIKE-A in a variety of human and that its activation of Akt to the of these PIKE-S and PIKE-L the GTPase domain that binds to these which we have directly not Overexpression of PIKE-A but not PIKE-S or PIKE-L in that form of PIKE is to growth Overexpression of PIKE-A but not PIKE-L or in the of in the of PIKE-L and PIKE-S but not PIKE-A. of such is a known regulatory of in 1999; PubMed Scopus Google Scholar). The cyclin-dependent in regulating cell proliferation and the are by phosphorylation mediated by the cyclin-dependent and and the cyclin-dependent S. M. M. 2002; Google Scholar). of results in the activation of including for cell progression. Here, we show that CDK4 is with PIKE-A in human cancer cells and genes to through signaling Akt occurs in the and to the nucleus with growth are known to Akt in the its in the nucleus not PIKE-A is and nuclear C. Liu C. L. Liu M. Cell. 2003; 23: PubMed Scopus Google it a physiologic regulator of Akt in the nucleus as as PIKE-S was originally identified as a PI 3-kinase demonstrate that PIKE-A not or PI 3-kinase of Akt by the GTPase PIKE-A an signaling pathway to the physiological role of Akt in independent of PI 3-kinase This is by that knockdown PIKE-A markedly Akt phosphorylation that the of PIKE-A with Akt and of Akt kinase activity the GTPase domain of PIKE-A, we have not an for In the of guanine wild-type PIKE-A binds to activated Akt but not Akt the dominant-negative PIKE-A binds to the active and Akt as a dominant-negative The with Akt of its phosphorylation in its phosphorylation in the regulatory and catalytic and Akt in the and and physiological GTPase and and to downstream PIKE-A binds to Akt in and shown that with GTPase with the and C. Xiao B. K. Nature. 2001; PubMed Scopus Google Scholar, J. FEBS Lett. 2001; PubMed Scopus Google Scholar). The as an the with Akt from but not from in the of guanine phosphorylation of Akt elicits leading to the binding of Akt for PIKE-A. This is by the interaction PIKE-A and with and B and the N-terminal GTPase domain and C-terminal ankyrin domain of PIKE-A are in with the regulatory and catalytic domains of Akt and PIKE-A binds active Akt in the of guanine its N-terminal the GTPase domain binds active and Akt in a in the PIKE-A protein, the binding domains from Akt in the of guanine to or the or of PIKE-A for active Akt because of the the binding domain in the (amino acids constitutively This is by the that the of to to active it contains the ankyrin repeats domain N-terminal GTPase and C-terminal ankyrin domains to the GTPase domain stimulates active and Akt kinase activity it is the GTPase domain that for the kinase to wild-type PIKE-A phosphorylation of Akt on and with a in in Akt kinase activity. the dominant-negative PIKE-A phosphorylation of Akt and its kinase activity The of PIKE-A regulates Akt to The is that wild-type PIKE-A binds Akt on the where and are the and it from protein that the Akt is dominant-negative PIKE-A with active and Akt from but the to which Akt activity is D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, J. M. M. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. P. 2002; PubMed Scopus Google Scholar, D.P. S. E. M. Hemmings B.A. 2001; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, H. H. D. B. B. Neuron. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, L. S. C. J. 2002; 21: PubMed Scopus Google Scholar, C. B.A. S. L. R. K. K. D.J. 1999; PubMed Scopus Google Scholar, A. C. R. M. S. P. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Our that growth factor the Akt to with PIKE-A in a guanine nucleotide-dependent way that GTPase PIKE-A a growth and cellular Akt. with dominant-negative PIKE-A that PIKE-A the physiologic activity of Akt as as for its to growth factor We and and for

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation. RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.