Hyperosmotic-induced Protein Kinase N 1 Activation in a Vesicular Compartment Is Dependent upon Rac1 and 3-Phosphoinositide-dependent Kinase 1

Abstract

Protein kinase N 1 (PKN1), which in part resembles yeast protein kinase C, has been shown to be under the control of Rho GTPases and 3-phosphoinositide-dependent kinase 1 (PDK1). We show here that green fluorescent protein-tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation, and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation is dependent on Rac1 activation, although the GTPase binding HR1abc domain is not sufficient for this recruitment. The PKN1 kinase domain, however, localizes constitutively to this compartment, and we demonstrate that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1-positive compartment upon hyperosmotic stress. Taken together, our findings present a pathway for the selective hyperosmotic-induced Rac1-dependent PKN1 translocation and PDK1-dependent activation. Protein kinase N 1 (PKN1), which in part resembles yeast protein kinase C, has been shown to be under the control of Rho GTPases and 3-phosphoinositide-dependent kinase 1 (PDK1). We show here that green fluorescent protein-tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation, and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation is dependent on Rac1 activation, although the GTPase binding HR1abc domain is not sufficient for this recruitment. The PKN1 kinase domain, however, localizes constitutively to this compartment, and we demonstrate that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1-positive compartment upon hyperosmotic stress. Taken together, our findings present a pathway for the selective hyperosmotic-induced Rac1-dependent PKN1 translocation and PDK1-dependent activation. Hyperosmotic stress is established as a potent activator of several signaling cascades, including stress-activated protein kinases (1Galcheva-Gargova Z. Derijard B. Wu I.H. Davis R.J. Science. 1994; 265: 806-808Crossref PubMed Scopus (531) Google Scholar), p38 (2Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar), and extracellular signal-regulated kinases (3Matsuda S. Kawasaki H. Moriguchi T. Gotoh Y. Nishida E. J. Biol. Chem. 1995; 270: 12781-12786Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In contrast, protein kinase B (PKB) 1The abbreviations used are: PKB, protein kinase B; PKN, protein kinase N; PI, phosphatidylinositol; PKC, protein kinase C; GFP, green fluorescent protein; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MBP, myelin basic protein; PAK, p21-activated kinase; GST, glutathione S-transferase; PDK1, 3-phosphoinositide-dependent kinase 1; GTPγS, guanosine 5′-3-O-(thio)triphosphate. is shown to be down-regulated by hyperosmotic stress via dephosphorylation of its regulatory Thr-308 and Ser-473 phosphorylation sites (4Meier R. Thelen M. Hemmings B.A. EMBO J. 1998; 17: 7294-7303Crossref PubMed Scopus (149) Google Scholar). Interestingly, the yeast PKC homologues, which in part resemble the PKNs, have been established as essential for the maintenance of cell wall integrity (5Arellano M. Valdivieso M.H. Calonge T.M. Coll P.M. Duran A. Perez P. J. Cell Sci. 1999; 112: 3569-3578Crossref PubMed Google Scholar). The regulation of cell wall integrity has been likened to the hyperosmotic stress response in Saccharomyces cerevisiae and has suggested roles for PKCs in high osmolarity responses (6Alonso-Monge R. Real E. Wojda I. Bebelman J.P. Mager W.H. Siderius M. Mol. Microbiol. 2001; 41: 717-730Crossref PubMed Scopus (76) Google Scholar). Notably, Mkh1, a yeast MEK kinase, is also shown to be activated and indeed required for cell wall integrity and a normal response to osmotic stress in Schizosaccharomyces pombe, further establishing the link between cell wall integrity and the osmotic stress response (7Sengar A.S. Markley N.A. Marini N.J. Young D. Mol. Cell. Biol. 1997; 17: 3508-3519Crossref PubMed Scopus (62) Google Scholar). Classical and novel PKC activation is suggested to be a requirement for hyperosmotic induced extracellular signal-regulated kinase activation (8Zhuang S. Hirai S.I. Ohno S. Am. J. Physiol. 2000; 278: C102—C109Crossref Google Scholar). This investigation has sought to address the involvement of PKN1 in the hyperosmotic stress response in mammalian cells. PKNs (protein kinase novel, also known as PRKs 2Here the nomenclature PKN is used throughout. PRK1 is referred to as PKN1, and PRK2 is referred to as PKN2. (9Mukai H. Ono Y. Biochem. Biophys. Res. Commun. 1994; 199: 897-904Crossref PubMed Scopus (140) Google Scholar, 10Palmer R.H. Ridden J. Parker P.J. FEBS Lett. 1994; 356: 5-8Crossref PubMed Scopus (40) Google Scholar)) are a subfamily of serine/threonine kinases identified independently by molecular cloning, protein purification, and PCR-based screens for PKC related kinases. The carboxyl-terminal kinase domains of these proteins are closely related to those of PKC, and at their amino termini they have a conserved repeated domain (HR1a,b,c) followed by a C2-related domain; overall these proteins have a domain organization related to that of the yeast PKC-related proteins (11Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1361) Google Scholar). PKNs are activated by fatty acids and phospholipids in vitro, although the in vivo significance of this remains unclear (12Morrice N.A. Fecondo J. Wettenhall R.E. FEBS Lett. 1994; 351: 171-175Crossref PubMed Scopus (19) Google Scholar, 13Palmer R.H. Dekker L.V. Woscholski R. Le Good J.A. Gigg R. Parker P.J. J. Biol. Chem. 1995; 270: 22412-22416Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The amino-terminal HR1 domain was identified as a Rho interacting region (14Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (350) Google Scholar, 15Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (396) Google Scholar, 16Vincent S. Settleman J. Mol. Cell. Biol. 1997; 17: 2247-2256Crossref PubMed Scopus (176) Google Scholar), and RhoB has been shown to target PKN1 to an endosomal compartment where it is implicated in controlling the kinetics of epidermal growth factor receptor traffic (17Mellor H. Flynn P. Nobes C.D. Hall A. Parker P.J. J. Biol. Chem. 1998; 273: 4811-4814Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18Gampel A. Parker P.J. Mellor H. Curr. Biol. 1999; 9: 955-958Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The interaction of Rho with PKN1 has been demonstrated to facilitate PKN1 activation loop phosphorylation by 3-phosphoinositide-dependent kinase 1 (PDK1). PDK1 was originally purified as an activity responsible for PKBα activation loop phosphorylation (19Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 20Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (916) Google Scholar). PDK1 has been demonstrated more recently to phosphorylate equivalent residues on many other AGC kinases, including p70S6k, cAMP-dependent protein kinase, and PKCs (reviewed in Ref. 21Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 3: 561-576Crossref Scopus (1400) Google Scholar). Based upon co-transfection experiments, the in vivo ternary complex of Rho-PKN1-PDK1 has been shown to be dependent on PI 3-kinase activity and to be critical for the catalytic activation of PKN1 (22Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). PKN1 has been linked to stress-induced pathways because it has been implicated upstream of c-Jun transcription via p38γ (23Marinissen M.J. Chiariello M. Gutkind J.S. Genes Dev. 2001; 15: 535-553Crossref PubMed Scopus (149) Google Scholar), both are activated upon hyperosmotic stress. Another relevant PKN response involves Fyn tyrosine kinase, which has been shown to mediate PKN2 function in keratinocytes (24Calautti E. Grossi M. Mammucari C. Aoyama Y. Pirro M. Ono Y. Li J. Dotto G.P. J. Cell Biol. 2002; 156: 137-148Crossref PubMed Scopus (146) Google Scholar) and has recently been shown (25Ko B.C. Lam A.K. Kapus A. Fan L. Chung S.K. Chung S.S. J. Biol. Chem. 2002; 277: 46085-46092Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) to be essential in transcription from the osmotic response element. Here we describe the acute translocation of GFP-PKN1 to vesicles in response to hyperosmotic stress. It is established that PKN1 translocation is dependent on Rac1 activation, and the kinase domain of PKN1 is shown to be an essential component of this response. The activity of PKN1 is found not to be required for vesicle recruitment, although activation loop phosphorylation and catalytic activation of PKN1 is shown to increase upon hyperosmotic stress. This activation is catalyzed by PDK1, which is recruited to the PKN1 compartment in a PI 3-kinase- and PKN1-dependent manner. Thus a pathway is established that leads from the plasma membrane activation of Rac1 to the vesicular accumulation of an activated PKN1 complex. from and the was as (22Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). the was from was from and was from The Rac1 activation was as a from used for was from and from The PI 3-kinase inhibitor and the inhibitor HA1077 from and by as PKN1 and PKN2 PKN1 was for PKN2 and 1 and a and and a The the PKN and This was with and and the of an amino-terminal with the PKN1 and PKN2. The PKN1 was in the manner as the from has been (17Mellor H. Flynn P. Nobes C.D. Hall A. Parker P.J. J. Biol. Chem. 1998; 273: 4811-4814Abstract Full Text Full Text PDF PubMed Scopus (100) Google was as J. R. Parker P.J. EMBO J. 2002; PubMed Scopus Google Scholar), and the kinase domain was from the The PKN1 kinase domain was from the a was by the from the kinase domain the sites of the was a from and the Rac1 and from Cell and in and with The was for to the and and Hyperosmotic to hyperosmotic for by the of and experiments, for and was with The of vesicles of hyperosmotic stress and and of was GFP-PKN1 as the of the and the of vesicles from was on and and as in the and in for for with for and in for with for 1 and with fluorescent for 1 and with the in and in on under and a with with and with and of and in to a Rac1 and in and for with to of hyperosmotic stress as in the The activation was on cell in the from This the binding domain of p21-activated kinase 1 as a protein that in a the of activated and to the was by and of on with GFP-PKN1 and to hyperosmotic stress as in of and on a loop phosphorylation of PKN1 was by a with of as (22Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). PKN1 phosphorylation was as a function of PKN1 protein the PKN1 GFP-PKN1 phosphorylation is from experiments, where the the GFP-PKN1 a of was with GFP-PKN1 and with hyperosmotic as in the in a CHAPS, 1 inhibitor from of of cell with protein was followed by with of for at by for 1 with protein G. with with and a in of GFP-PKN1 was in a of of of myelin basic protein and 1 of for at with by the of of to on a and was from to to with activity was as a function of The are of experiments, and where the HA1077 PKN as a control for PKN activity M. Chihara K. N. T. Y. Kaibuchi K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. Cohen P. Biochem. J. 2000; 351: PubMed Scopus Google Scholar). GFP-PKN1 in a with an of The of a of PKN1 on the of GFP-PKN1 was including the epidermal growth growth growth and stress as and hyperosmotic stress. Notably, with hyperosmotic for a in with GFP-PKN1 in vesicular The of the hyperosmotic-induced was by following of hyperosmotic stress. GFP-PKN1 vesicles and by vesicles on from hyperosmotic that the of vesicles cell in a of It is that the accumulation of vesicular PKN1 is a reversible has been shown to in vesicular following PKC in J. R. Parker P.J. EMBO J. 2002; PubMed Scopus Google Scholar). the of the PKN1 translocation in response to hyperosmotic PKN1 and has been with GFP-PKN1 to that they show the behavior and hyperosmotic stress not The ability of PKN1 to translocate vesicles upon hyperosmotic stress was by the of not to the that the behavior of PKN1 in response to hyperosmotic stress The ability of other PKN subfamily to to hyperosmotic stress was also was with accumulation hyperosmotic the translocation as GFP-PKN1 that the behavior is a PKN response. The of catalytic activity on this was by the which has been shown to both PKN1 and PKN2 M. Chihara K. N. T. Y. Kaibuchi K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. Cohen P. Biochem. J. 2000; 351: PubMed Scopus Google Scholar). of with HA1077 followed by hyperosmotic stress not PKN1 vesicle recruitment. with HA1077 an accumulation of vesicular GFP-PKN1 further the of PKN1 we the this PKN1 a and hyperosmotic stress in vesicles as for and to of PKN1 in a with to vesicle recruitment. and PKN1 in and in vesicles upon hyperosmotic stress findings that the kinase activity of PKN1 is not necessary for vesicle recruitment. the of HA1077 in PKN1 a in the from this vesicular compartment, which be part of a pathway and PKN has been shown to to and activated by of the Rho of GTPases via the regulatory HR1 domain and indeed has been shown to be recruited to an endosomal compartment by RhoB (17Mellor H. Flynn P. Nobes C.D. Hall A. Parker P.J. J. Biol. Chem. 1998; 273: 4811-4814Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We the involvement of Rho proteins in the PKN response to hyperosmotic stress by the from has been shown to the Rho subfamily other Rho GTPases as Rac1 C. H. K. I. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). with GFP-PKN1 and with for at followed by to hyperosmotic stress. The of cell was by an in and also by the of stress by not The accumulation of vesicles was of that Rho function is not required for this response not The GTPase Rac1 has been implicated in the control of PKN2 S. Settleman J. Mol. Cell. Biol. 1997; 17: 2247-2256Crossref PubMed Scopus (176) Google Scholar) and recently in response to hyperosmotic A. C. Kapus A. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). in the of a Rho the involvement of Rac1 in the translocation of PKN1 was Rac1 was with control GFP-PKN1 was and Rac1 to the plasma and to vesicular of hyperosmotic and GFP-PKN1 in vesicles of the with GFP-PKN1 in vesicular translocation of GFP-PKN1 hyperosmotic stress that of Rac1 is necessary for the hyperosmotic stress-induced of Rac1 has been shown recently to upon hyperosmotic stress in A. C. Kapus A. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). This was here by a Rac1 the domain of that of hyperosmotic a increase in the of Rac1 was to the HR1 domain of PKN proteins P. Mellor H. R. G. Parker P.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus the of the regulatory HR1abc region on hyperosmotic stress-induced vesicle was of the HR1abc domain of This domain the cell under control and hyperosmotic stress not its was with GFP-PKN1 to their The of the HR1abc domain with GFP-PKN1 hyperosmotic stress. The of HR1abc not GFP-PKN1 vesicle The that Rac1 PKN1 at the plasma membrane where HR1abc for interaction and that the complex not the complex is We that because HR1abc was to GFP-PKN1 vesicle Rac1 remains in under these GFP-PKN1 for Rac1 in the of of the of the between these the behavior of the HR1abc domain that although the regulatory of Rac1 PKN1 be essential in PKN1 for recruitment, it be the by which PKN1 vesicle is the of other PKN domains in vesicle recruitment, the kinase domain was cells. control kinase domain a vesicular and upon hyperosmotic this vesicular more the kinase domain to the as the the kinase domain was with hyperosmotic stress they found to in vesicular The that the PKN1 kinase domain by to vesicles the that the kinase domains of related PKC in a manner. kinase domain was with the kinase hyperosmotic stress the kinase domain of PKN1 be in in these the kinase domain of was that vesicle of PKN1 the kinase domain is for this compartment and with a closely related kinase The HA1077 on the and osmotic induced of PKN1 that its activity be required for vesicle it is not activation of PKN in response to osmotic stress. loop phosphorylation is required for catalytic activity of PKN1 (22Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). used to the of hyperosmotic on GFP-PKN1 activation loop increase in phosphorylation of hyperosmotic stress was on GFP-PKN1 We also to an increase in PKN1 phosphorylation hyperosmotic GFP-PKN1 from to was used to the of osmotic stress on hyperosmotic GFP-PKN1 a activity GFP-PKN1 The of kinases responsible for this activity was with control the PKN inhibitor This inhibitor has been to be for PKN1 kinases that with it PDK1, M. Chihara K. N. T. Y. Kaibuchi K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. Cohen P. Biochem. J. 2000; 351: PubMed Scopus Google Scholar). GFP-PKN1 activity hyperosmotic was to in the of PDK1 has been shown to to and facilitate the activation loop phosphorylation of PKN1 (22Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). the that PKN1 is both and activated osmotic we the involvement of PDK1 in the control of vesicular was with The of both proteins was in hyperosmotic and found to be in vesicles PI 3-kinase PDK1 via its domain D.R. M. A. N. Gaffney P. Reese C.B. D. A. M. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Scopus Google Scholar, D. T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: PubMed Scopus Google Scholar) and the phosphorylation and activation of PKN1 (22Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). We the on osmotic responses following with the PI 3-kinase inhibitor hyperosmotic was recruited to Notably, was recruited to however, these the with The here demonstrate that PKN1 is by hyperosmotic the of a vesicular complex with the upstream kinase This is by the activation of although this is found to be for accumulation of vesicular of interaction between PKN1 and the compartment to be and this is with the that the kinase domain is to Based upon of an PKN1 it is shown that PKN1 catalytic activity is not required for vesicle recruitment, a by the vesicular accumulation of PKN1 in the of the catalytic inhibitor the of PDK1 leads to the activation loop phosphorylation of PKN1 that its catalytic activation. A. C. Kapus A. Am. J. Physiol. 2002; PubMed Scopus Google Scholar) have the of Rac1 in response to hyperosmotic a here in a cell In fact a hyperosmotic stress response has been for Rac1 p21-activated protein kinase which to and is activated by has been shown to translocate from a to a and activated in response to J. Z. C. J.A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, it was demonstrated that the activation not translocation of was to a for the response to hyperosmotic stress. This the here for PKN1 where of PI 3-kinase not vesicle accumulation of PKN1, of the upstream kinase It has been shown (22Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) that of PI 3-kinase activation loop phosphorylation of The of the induced PKN1 vesicular compartment here is not that this is not an endosomal compartment an compartment not the of PI 3-kinase on the induced PKN1-positive compartment be with this part of an where of PI 3-kinase M.J. Biochem. J. 1995; PubMed Scopus Google Scholar). Based upon with the factor it is that interaction is not required for the of PKN1 in response to hyperosmotic because Rac1 has also been to the HR1abc domain of PKN1 and also because Rac1 in response to hyperosmotic the of this PKN was The ability of the Rac1 to PKN accumulation in the vesicular compartment that Rac1 a in this hyperosmotic induced this is for because the HR1abc domain of PKN1 is not recruited to vesicles the Rac1 interacting domain (14Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (350) Google Scholar, 15Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (396) Google Scholar, 16Vincent S. Settleman J. Mol. Cell. Biol. 1997; 17: 2247-2256Crossref PubMed Scopus (176) Google Scholar). The that the PKN1 kinase domain is in part constitutively with the that PKN1 Rac1 activation, that Rac1 binding to PKN1 on hyperosmotic stress a the catalytic domain and vesicular recruitment. with it has been suggested that the interaction of Rho GTPases at the amino-terminal HR1 to an interaction activation, an M. H. M. Mukai H. Ono Y. Biochem. Biophys. Res. Commun. 1996; PubMed Scopus Google Scholar). The of with PKN1 has been shown to increase the phosphorylation and catalytic activity of PKN1 (14Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (350) Google Scholar, 15Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (396) Google Scholar, 16Vincent S. Settleman J. Mol. Cell. Biol. 1997; 17: 2247-2256Crossref PubMed Scopus (176) Google Scholar), and the behavior of PKN1 here the that the the amino-terminal HR1 domain is required for complex with and phosphorylation by The of PDK1 the kinase domain not and is to the as a D. M. Alessi D.R. EMBO J. 2002; PubMed Scopus Google Scholar). This vesicular interaction is not responsible for the accumulation of PKN1, because this PDK1 is by the 3-kinase inhibitor Thus the kinase domain in the vesicle membrane be by a protein The of on PDK1 to PKN1 that is required to the of PKN1 that of PDK1 to facilitate complex of these proteins have been shown to be by however, in the of PKN1 was to the R.H. Dekker L.V. Woscholski R. Le Good J.A. Gigg R. Parker P.J. J. Biol. Chem. 1995; 270: 22412-22416Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). it is that the of is to membrane of PDK1 its domain D.R. M. A. N. Gaffney P. Reese C.B. D. A. M. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Scopus Google Scholar) and facilitate to the The in of PKN1-positive vesicles on of with that the vesicles are a of a PI vesicle this is a of M.J. Biochem. J. 1995; PubMed Scopus Google Scholar). The regulatory that PDK1 phosphorylation of PKN1 further for the that the of PDK1 is by the of regulatory to target kinases B. Alessi D.R. Biochem. J. 2000; 3: 561-576Crossref Scopus (1400) Google Scholar). In this it is that the requirement of PI 3-kinase for PDK1 recruitment, PKB, which is also recruited by is not recruited to this hyperosmotic induced endosomal compartment not This that is that under other are required for PDK1 phosphorylation of is a known Am. J. Physiol. 1998; 275: Scholar, A. P. G. S. Biochem. Biophys. Res. Commun. 2000; 270: PubMed Scopus Google Scholar), and PKNs have been shown to undergo in response to and under Y. A. Mellor H. L. Parker P.J. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. N. T. C. M. M. Ono Y. Saito N. Sci. 2000; 41: Google Scholar). we have not of PKN1 under osmotic stress. The responses here that in fact the behavior of PKN1 under hyperosmotic an The vesicular of the kinase domain in the of hyperosmotic and the that HA1077 PKN1 vesicle accumulation that this is a pathway by hyperosmotic The findings that the PKN1 response to is not PKN1 for that PKN1 activity is in the from this vesicular In we describe PKN1 to be a kinase, with the induced translocation selective related although with PKN2. stress induced signaling are stress although upon investigation of other as and PKN1 translocation was not We that the PKN1 response is for hyperosmotic stress. The selective translocation of PKN1 with its activation a by which hyperosmotic a of responses this We for the We also Alessi for the PDK1 Hall for the Rac1 and for