Oncogene (1999) 18, 8024 ± 8032 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism Danielle K Lynch1, Christine A Ellis2, Paul AW Edwards1 and Ian D Hiles*,2 1Department of Pathology, Cambridge Unversity, Tennis Court Road, Cambridge CB2 1QP, UK; 2Molecular Pharmacology Unit, GlaxoWellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK The serine threonine kinase protein kinase B regulates Kitamura et al., 1998; Ueki et al., 1998). In contrast, cellular activities as diverse as glycogen metabolism and insulin-like growth factor-1 treatment of COS7 cells apoptosis. Full activation of protein kinase B requires 3- and nerve growth factor-dependent neurons confers phosphoinositides and dual phosphorylation on threonine- PKB-mediated protection from apoptosis following 308 and serine-473. CaM-K kinase and 3-phosphoinosi- UV treatment and serum withdrawal, respectively tide dependent-kinase-1 phosphorylate threonine-308. (Kulik et al., 1997; Dudek et al., 1997). PKB Integrin-linked kinase reportedly phophorylates serine- activation also protects myc transfected Rat-1 fibro- 473. Consistent with this, in a model COS cell system we blasts (Kauman-Zeh et al., 1997) and MDCK show that expression of wild-type integrin-linked kinase epithelial cells (Khwaja et al., 1997) from the promotes the wortmannin sensitive phosphorylation of apoptosis (termed anoikis ± Frisch and Francis, 1994) serine-473 of protein kinase B and its downstream that normally accompanies cell detachment from substrates, and inhibits C2-ceramide induced apoptosis. extracellular matrix. PKB dependent phosphorylation In contrast, integrin-linked kinase mutated in a lysine of serine-136 (S136) of BAD, a protein involved in residue critical for function in protein kinases is inactive apoptotic signalling (Datta et al., 1997; Blume-Jensen in these experiments, and furthermore, acts dominantly et al., 1998; Chao and Korsmeyer, 1998), leads to its to block serine-473 phosphorylation induced by ErbB4. inactivation and may contribute to the regulation of However, alignment of analogous sequences from apoptosis by PKB. Direct phosphorylation and dierent species demonstrates that integrin-linked kinase inhibition of both caspase 9 and the forkhead is not a typical protein kinase and identi®es a conserved transcription factor FKHLR1 by PKB are also likely serine residue which potentially regulates kinase activity to be important in the regulation of apoptosis in a phosphorylation dependent manner. Mutation of this (Cardone et al., 1998; Brunet et al., 1999). PKB is serine to aspartate or glutamate, but not alanine, in also activated by integrins (Frisch and Ruoslahti, 1997; combination with the inactivating lysine mutation King et al., 1997; Dimmeler et al., 1998; Guilherme restores integrin-linked kinase dependent phosphorylation and Czech, 1998) and the dual regulation of PKB by of serine-473 of protein kinase B. These data strongly growth factors and integrins suggests that PKB suggest that integrin-linked kinase does not possess activation represents a point where signals from serine-473 kinase activity but functions as an adaptor growth factors and extracellular matrix converge in a to recruit a serine-473 kinase or phosphatase. cell. Structurally, PKB possesses an N-terminal pleckstrin Keywords: Akt; ErbB4; integrin-linked kinase; phos- homology (PH) domain that binds lipid products of phatidylinositol 3-kinase; protein kinase B; site-directed phosphatidylinositol 3-kinase (PI3-kinase), a central mutagenesis kinase domain and a c-terminal extension containing phosphorylation sites (Marte and Downward, 1997; Coer et al., 1998). The regulation of PKB kinase activity is complex and involves several distinct steps Introduction (Bellacosa et al., 1998; Coer et al., 1998). The PH domain of PKB binds to 3-phosphoinositide products Protein kinase B (PKB), the cellular homologue of the of PI3-kinase and this interaction is implicated in both viral oncogene v-akt, is a serine-threonine protein membrane targeting and kinase activation (Anjelkovic kinase (Bellacosa et al., 1991; Coer and Woodgett, et al., 1997; Klippel et al., 1997; Franke et al., 1997). 1991; Jones et al., 1991). PKB regulates many diverse Dual phosphorylation on both threonine-308 (T308), pathways and responses in a cell, depending on cell within the activation loop of the kinase domain, and type (reviewed in Marte and Downward, 1997; Coer serine-473 (S473) in the c-terminal tail is also required et al., 1998). For example, insulin stimulation of L6 for full activation of kinase activity (Alessi et al., 1996). muscle cells results in PKB-dependent phosphorylation A protein kinase that phosphorylates T308 has been and inhibition of glycogen synthase kinase 3a (GSK- characterized and termed 3-phosphoinositide depen- 3a) (Cross et al., 1995; van Weeren et al., 1998) and dent-kinase 1 (PDK1) (Alessi et al., 1997a, b; Cohen et stimulation of protein synthesis (Gingras et al., 1998; al., 1997; Stephens et al., 1998). Like PKB, PDK1 also contains a PH domain that binds the lipid products of PI3-kinase (Alessi et al., 1997b; Stephens et al., 1998). Phosphorylation of T308 in vivo is dependent on PI3- *Correspondence: I Hiles Received 23 June 1999; revised 27 September 1999; accepted kinase activity, but it is unclear if this requirement is 28 September 1999 necessary for the unfolding of PKB to allow access of Regulation of protein kinase B by ILK DK Lynch et al 8025 PDK1 to T308, direct activation of PDK1 through its methionine (Carrera et al., 1993; Hanks and Hunter, PH domain, or both of these events (Bellacosa et al., 1995). Figure 1a, lane 5, shows that co-expression of 1998; Stephens et al., 1998). Ca2+/calmodulin-depen- ILK and ErbB4 CYT-1 in serum starved COS cells dent protein kinase kinase (CaM-KK) can also augments phosphorylation of S473 or PKB. Indeed, phosphorylate T308 or PKB in response to calcium enhanced phosphorylation of PKB on S473 is notice- (Yano et al., 1998). The PI3-kinase dependent kinase able in cells expressing ILK protein alone in the activity that phosphorylates S473 or PKB in vivo, absence of ErbB4 CYT-1 (for example compare lanes 1 which has been termed 3-phosphoinositide dependent kinase 2 (PDK2) (Alessi et al., 1997a), is less well characterized. MAPKAP kinase 2 is capable of phosphorylating S473 in vitro, but is unlikely to be an important in vivo regulator of PKB (Alessi et al., 1996). In vitro, PDK1 bound to protein kinase-C related kinase-2 (PRK2) or a c-terminal peptide derived from PRK2 can phosphorylate S473 of PKB (Balendran et al., 1999). However it is unclear if this PDK1/PRK2 complex phosphorylates S473 in vivo.A third candidate for a S473 kinase is integrin-linked kinase (ILK) (Delcommenne et al., 1998), originally identi®ed as a b1-integrin binding protein in a yeast 2- hybrid screen (Hannigan et al., 1996). However, the sequence of the kinase domain of ILK diers from the consensus for a protein kinase in several residues considered critical for kinase activity. In this paper, we assess ILK S473 kinase activity in cells. Expression of exogenous ILK promotes phos- phorylation of S473 of PKB resulting in phosphoryla- tion of downstream eectors of PKB and inhibition of C2-ceramide induced apoptosis. Using sequence align- ment of ILK analogues from dierent species combined with site-directed mutagenesis of ILK, we probe how ILK functions as a S473 kinase. We conclude that ILK regulates S473 phosphorylation by acting as an adaptor, either to recruit a distinct S473 kinase activity or to inhibit a S473 speci®c phosphatase activity. Results ILK regulates S473 phosphorylation of PKB A COS cell transient-expression system based on expression of 2 isoforms of ErbB4 (CYT-1 and CYT- 2) was established to study signalling pathways dependent on PI3-kinase activation. CYT-1 and CYT-2 dier by the presence or absence of 16 amino acids containing a consensus binding site for PI3- kinase (Sawyer et al., 1998; Elenius et al., 1999). As expected, the longer isoform CYT-1 (containing the PI3-kinase binding site), in contrast to CYT-2, promoted the recruitment and activation of PI3-kinase (data not shown) and this correlated with increased phosphorylation of both S473 and T308 of PKB Figure 1 Eect of integrin-linked kinase on phosphorylation of (Figure 1a, compare lanes 4 and 7, Ser473 and protein kinase B, glycogen synthase kinase-3a, BAD and MAP- kinase in serum starved transiently transfected COS cells. COS Thr308 panels). In all other respects, CYT-1 and cells were transiently transfected with ErbB4 CYT-1, lanes 4, 5 CYT-2 behaved identically. Both forms associated and 6, ErbB4 CYT-2, lanes 7, 8 and 9 or mock transfected (COS), equally well with SHC, NCK and GRB2 and lanes 1, 2 and 3. Certain COS cells were additionally transfected stimulated MAP-kinase activity (data not shown and with integrin-linked kinase (WT-ILK) (lanes 2, 5 and 8) or a mutated integrin-linked kinase (DN-ILK) (lanes 3, 6 and 9). Figure 1c lanes 4 and 7). RIPA buer extracts were fractionated by SDS ± PAGE, blotted Using the COS cell system, we investigated the to PVDF membrane and probed with antisera speci®c for; (a) ability of ILK or a mutant of ILK (DN-ILK) to ILK, ErbB4, PKB, phospho ser473 PKB, phospho thr308 PKB, promote phosphorylation of S473 of PKB. DN-ILK (b) GSK3 (a and b forms), phospho Ser21 GSK-3a, BAD or was generated by changing a critical lysine residue phospho Ser136 BAD, (c) MAP kinase or phospho MAP kinase as indicated. Where indicated, cells were treated with 100 nM (lysine 220 in ILK ± see Figure 3a) conserved in most wortmannin for 30 min prior to cell lysis.