Oncogene (1999) 18, 8024 ± 8032 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc -linked kinase regulates phosphorylation of serine 473 of 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 -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 (Kau€man-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. . 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 di€erent 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 (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; Co€er et al., 1998). The regulation of PKB kinase activity is complex and involves several distinct steps Introduction (Bellacosa et al., 1998; Co€er 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; Co€er 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; Co€er 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 di€ers 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 e€ectors of PKB and inhibition of

C2-ceramide induced apoptosis. Using sequence align- ment of ILK analogues from di€erent 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 di€er 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 E€ect 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 bu€er 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. Equal amounts of protein kinases, and essential for kinase activity, to protein were loaded per track Regulation of protein kinase B by ILK DK Lynch et al 8026

Figure 2 E€ects of integrin-linked kinase expression on apoptosis induced by C2-ceramide in COS cells. COS cells were transiently transfected with WT-ILK, DN-ILK or vector alone. Cells were serum starved overnight and treated with 50 mM C2-ceramide (Cera) or C2-dihydroceramide (DHC) or ethanol vehicle for 8 h and the percentage of cells undergoing apoptosis determined by FACS analysis (a) or TUNEL (b). Numbers in (a) and (b) represent the average of ®ve and three experiments respectively, each performed in triplicate. Error bars represent standard deviation. (c) COS cells were serum starved overnight and treated with 50 mM C2- ceramide (C2-ceramide) or C2-dihydroceramide (C2 DHC) or ethanol vehicle (mock). Alternatively COS cells were transfected with WT-ILK or DN-ILK, serum starved overnight and treated with 50 mM C2-ceramide (WT-ILK+CER and DN-ILK+CER, respectively). After the indicated times, cell survival was calculated by staining with trypan blue. A minimum of 600 cells were counted per point, where possible. (d) COS cells were transiently transfected with WT-ILK, lanes 2, 5 and 8, DN-ILK, lanes 3, 6 and 9, or mock transfected, lanes 1, 4 and 7. Cells were serum starved overnight and treated with 50 mM C2-ceramide (Ceramide), lanes 4, 5 and 6, or C2-dihydroceramide (DHC), lanes 7, 8 and 9, or ethanol vehicle, lanes 1, 2 and 3, for 8 h. RIPA extracts were separated by SDS ± PAGE, transferred to PVDF membrane and blotted with antisera speci®c for PKB (top) and phospho-S473 PKB (bottom). Equal amounts of protein were loaded per track

and 2, and 7 and 8 in Figure 1a, S473 panel). that is una€ected by expression of ILK or DN-ILK Expression of DN-ILK leads to a drastic diminution (Figure 1a), changes in S473 phosphorylation induced of phosphorylation of S473 of PKB e.g. Figure 1a, by expression of ILK and ILK mutants should result compare lanes 4 and 6. Western blotting with an ILK- in parallel changes in PKB activity towards down- speci®c polyclonal antiserum con®rms expression of stream substrates. ILK and DN-ILK in transfected cells at levels Therefore, phosphorylation on two PKB substrate approximately ®vefold higher than that of endogenous sites, serine 21 of glycogen synthase kinase 3a (GSK- protein (Figure 1a, top). DN-ILK acts speci®cally to 3a) and serine 136 of BAD, was assessed using reduce S473 phosphorylation of PKB and not by a antibodies speci®c for phosphorylated forms of these general non-speci®c inhibition of kinase function, since proteins. As in previous studies (Blume-Jensen et al., it had no e€ect on the phosphorylation of MAP kinase 1998), co-transfection of a BAD expression plasmid (MAPK) (Figure 1c). Control blots (Figure 1a, PKB proved necessary to investigate phosphorylation of panel) show that changes in PKB expression levels do S136 of BAD as endogenous BAD levels in COS cells not account for the observed changes in S473 are very low. Figure 1b shows that expression of ILK phosphorylation. Treatment with the PI3-kinase enhances phosphorylation of S21 of GSK-3a (for inhibitor wortmannin (100 nM) (Ui et al., 1995) totally example, Figure 1b, phospho-S21 GSK-3a panel, abolishes the ILK-dependent increase in S473 phos- compare lanes 1 and 2, 4 and 5, 7 and 8). A similar phorylation consistent with ILK acting via a PI3- e€ect can also be seen on the phosphorylation of S136 kinase dependent mechanism (Figure 1a, bottom). of BAD (Figure 1b, phospho-S136 BAD panel, Activation of PKB requires dual phosphorylation on compare lanes 1 and 2, 4 and 5, and 7 and 8). For T308 and S473. Since T308 is constitutively phos- S136 of BAD there is a high basal level of phorylated in serum-starved COS cells at a basal level phosphorylation that was consistently increased two- Regulation of protein kinase B by ILK DK Lynch et al 8027

Figure 3 (a) An alignment of the human, Caenorhabditis elegans and ILK protein sequences, and human B-Raf (residues 445 ± 719) using Clusta1W (Thompson et al., 1994). #### indicates the position of the potential FSF phosphorylation site sequence in the activation loop of ILK. The three conserved ankyrin repeats are indicated. A* marks the position of the lysine residue changed to methionine to create DN-ILK. The DFG motif, only present in B-Raf, is underlined. (b) Calculation of percentage identi®es between the three ILK homologous sequences using 1Align (Huang and Miller, 1991). (c)An alignment of the predicted catalytic loop sequences of the three ILK sequences and B-Raf with the protein kinase consensus (Hanks and Hunter, 1995). A* marks the position of the catalytic aspartate residue critical for kinase function

fold by expression of ILK (measured by densitometry ± treatment (100 nM) abolished both the S21 phosphor- data not shown). S136 of BAD is phosphorylated by ylation of GSK-3a and the increase in the S136 other protein kinases and these are likely to contribute phosphorylation of BAD (Figure 1b). to the phosphorylation on this site seen in this experiment (Scheid and Duronio, 1998). Expression ILK expression rescues C -ceramide induced apoptosis of DN-ILK leads to signi®cant inhibition of phosphor- 2 ylation of S21 of GSK-3a and S136 of BAD (Figure Having demonstrated the ability of ILK to mediate 1b, compare lanes 4 and 6, phospho-21GSK-3a and phosphorylation of PKB and its substrates, we next phospho-S136 BAD panels, respectively). Wortmannin tested whether ILK expression modulates PKB Regulation of protein kinase B by ILK DK Lynch et al 8028

biological function. Treatment of COS cells with C2- residues should be conserved during evolution. Figure ceramide induces apoptosis and this can be inhibited 3a shows an alignment of these three ILK sequences by the activation of PKB (Zhou et al., 1998). We together with the B-Raf sequence as a reference kinase. examined the e€ects of expressing ILK or DN-ILK on The overall degree of sequence conservation (Figure the percentage of cells undergoing apoptosis in this 3b) suggests the predicted C. elegans and D.

assay. C2-ceramide treatment of cells caused several melanogaster proteins are likely to have homologous morphological changes indicative of apoptosis. These functions to human ILK. Near the N-terminus all three included, loss of viability, cell shrinkage, decreased ILK homologues possess three ankyrin repeats adherence to tissue culture treated plastics, cell shape (underlined in Figure 3a), followed by a linker region changes, the increased appearance of round phase that may bind lipids (Delcommenne et al., 1998) and bright cells and membrane blebbing (data not shown). then a kinase homology domain. A fourth ankyrin The proportion of apoptotic cells was quantitated by repeat has been noted in human ILK (Hannigan et al., two di€erent methods (1) FACS analysis of the sub-G1 1996) but the sequence identi®ed is divergent from the peak following staining with propidium iodide consensus for the ankyrin repeat motif and it is not (indicative of DNA fragmentation); (2) TUNEL (a conserved through evolution. measure of DNA fragmentation and strand breakage). Comparison of the kinase domain of ILK to that of In addition, trypan blue exclusion from the nucleus (a other protein kinases e.g. B-Raf (Figure 3a) shows that measure of the integrity of the nuclear membrane) was residues important in maintaining kinase structure are used to measure cell viability. Figures 2a (propidium conserved, but that residues involved in catalytic iodide staining) and 2b (TUNEL) show that markedly function are more divergent (Hubbard et al., 1994; increased apoptosis occurs in COS cells treated with Hanks and Hunter, 1995). In particular, ILK sequences

C2-ceramide but not the biologically inactive analogue, corresponding to the catalytic loop show little

C2-dihydroceramide. Cell viability is also decreased by similarity, implying they have not been constrained

C2-ceramide (Figure 2c). In all three assays, ILK by function during evolution. None of the ILK expression signi®cantly reduced the number of cells sequences have a DFG motif or a conserved substitute undergoing apoptosis/loss of viability following treat- for the catalytic aspartate residue found in other

ment with C2-ceramide (Figure 2a, b and c). Expression kinases. A phe-ser-phe (FSF) motif is conserved in all

of DN-ILK has no e€ect. Morphologically, C2- three ILK analogues within a sequence corresponding ceramide treated cells expressing exogenous ILK to the activation loop of protein kinases (Hubbard et

resemble those treated with C2-dihydroceramide or al., 1994; Hanks and Hunter, 1995 ± marked with ### ethanol vehicle alone, whereas those expressing DN- in Figure 3a. In many kinases including insulin ILK were rounded up, shrunken and loosely attached receptor, PKB and MAP kinase, residues in the to the dish (data not shown). The number of apoptotic activation loop are subject to reversible phosphoryla- events recorded by each method of assessment varies tions which control kinase activity (Her et al., 1993; owing to di€erences in sensitivity of the detection Hubbard et al., 1994; Co€er et al., 1998). Substitution method and the di€ering end points i.e. the stage of of a negatively charged residue e.g. aspartate or apoptosis measured by each technique. In each case, glutamate for the phosphorylated residue mimics the however, expression of ILK results in a twofold change in charge that accompanies phosphorylation

decrease in C2-ceramide induced-apoptosis compared and results in constitutive activation of the kinase. In to control treated cells and cells expressing DN-ILK contrast, substitution of alanine prevents phosphoryla- (Figure 2a and b). The extent of rescue we observe with tion consequently inhibiting kinase activation. FSF is ILK may be an underestimate of the real e€ect as our similar to the consensus sequence de®ned for transfection eciency of 50 ± 80% means that 80% phosphorylation by PDK2, phe-X-X-phe-ser-phe/tyr rescue rather than 100% is the maximal response we (Pearson et al., 1995; Alessi et al., 1996). If ILK could anticipate. Increased PKB activity also rescues possesses intrinsic PDK2 activity, this site may be

COS cells from C2-ceramide induced apoptosis (Zhou subject to autophosphorylation and possibly regulate et al., 1998). Since we have shown ILK regulates PKB ILK activity. To investigate this we changed the serine activity, we propose ILK regulates COS cell apoptosis residue of the FSF motif of either wild-type ILK or through a PKB-dependent pathway. Interestingly, ILK DN-ILK to alanine, aspartate or glutamate, and

induced S473 phosphorylation is reduced by C2- assessed the e€ect on the phosphorylation of S473 of ceramide treatment compared to treatment with the PKB.

inactive analogue C2-dihydroceramide (Figure 2d) suggesting in COS cells C -ceramide exerts its e€ects 2 E€ects of changes in the FSF motif on PKB-S473 by down-regulation of PKB activity. phosphorylation Figure 4 shows that expression of either FDF or FEF ILK is conserved through evolution and lacks key point-mutated ILK induce phosphorylation of S473 of catalytic residues PKB. In contrast ILK-FAF is inactive. These data Since ILK lacks both HRDLXXN (containing the support the idea that the charge on the serine residue catalytic aspartate residue) and DFG (involved in the of the FSF motif is important in regulating ILK coordination of a magnesium ion) motifs that are function and are consistent with this being a site for considered to be critical for kinase function. (Hannigan phosphorylation. Changes in expression levels of ILK et al., 1996; Hanks and Hunter, 1995), we were and PKB do not account for the large changes intrigued by the mechanism of ILK function. We observed in S473 phosphorylation (Figure 4, ILK and sequenced ILK analogues from C. elegans and D. PKB panels). DN-ILK-FAF was inactive in promoting melanogaster, arguing that functionally important S473 phosphorylation. However, surprisingly, both Regulation of protein kinase B by ILK DK Lynch et al 8029 DN-ILK-FDF and DN-ILK-FEF promote phosphor- Discussion ylation of S473 of PKB at a third of the wild type level, even though from many previous studies the lysine to Our results clearly demonstrate that ILK induces methionine mutation is expected to eciently inhibit phosphorylation of S473 of PKB in COS cells. PKB protein-kinase activity (Figure 4, S473 panel) (Carrera S473 phosphorylation correlates with the phosphoryla- et al., 1993; Hanks and Hunter, 1995). None of the tion of PKB substrates, GSK3a and BAD, and the mutations had a signi®cant e€ect on the phosphoryla- inhibition of apoptosis induced by treatment of COS tion state of T308 (Figure 4). We next examined the cells with C2-ceramide. Interestingly, the same apparent ability of the PI3-kinase inhibitor wortmannin to degree of phosphorylation of S473 on PKB leads to inhibit the phosphorylation of S473 of PKB induced di€erent e€ects on substrate phosphorylation depend- by expression of the various mutant ILK proteins. ing on whether ErbB4 is expressed or not. For Surprisingly, S473 phosphorylation induced by expres- example, in Figure 1a and b, the extent of phosphor- sion of ILK-FDF, ILK-FEF, DN-ILK-FDF and DN- ylation of S473 of PKB is similar in lanes 2 and 8, but ILK-FEF was partially resistant to wortmannin the phosphorylation on S21 of GSK-3a is reduced in treatment at concentrations that completely abolished lanes 8 compared to lane 2. Similarly, co-expression of S473 phosphorylation promoted by wild type ILK DN-ILK and CYT-1 results in a large but partial loss (Figure 4, bottom). In all cases phosphorylation on S21 of S473 phosphorylation of PKB, yet a complete loss of the PKB substrate GSK3-a was observed to change of phosphorylation of S21 of GSK-3a (Figure 1a and in parallel with changes in phosphorylation of S473 of b, lane 6, phospho S473 PKB and phospho S21 GSK- PKB (data not shown). Thus by substituting a 3a blots). Although the reason for this is unclear, other negatively charged residue in place of the serine kinases such as PKC can also phosphorylate S21 of residue of the FSF motif, we have changed the ILK GSK-3a (Cook et al., 1996). Constitutive activation of induced-phosphorylation of S473 of PKB from a PI3- ErbB4 could result in down-regulation of the activity kinase dependent, to a partially PI3-kinase independent of these kinases or up-regulation of an S21 phospha- mechanism. Furthermore, substitution of FEF or FDF tase activity. Delcommenne et al., 1998 concluded it in the DN-ILK background restores function to the most likely that ILK directly regulates S473 phosphor- otherwise defective DN-ILK. This ability to restore ylation of PKB. However, our data suggest this is activity to DN-ILK argues strongly against ILK being improbable for three reasons. (1) We can detect no PDK2. signi®cant protein kinase activity in immuneprecipi- tates of ILK using a variety of di€erent substrates, including PKB (data not shown). Others have also failed to detect kinase activity in ILK immuneprecipi- tates (Balendran et al., 1999). (2) ILK lacks both a DFG motif and the catalytic aspartate residue considered essential for ecient catalytic activity (Figure 3). In protein kinase A (PKA), mutation of the catalytic aspartate residue to alanine results in a signi®cant reduction of catalysis to 0.4% of wild type activity (Gibbs and Zoller, 1991). Thus, although ILK may possess a low intrinsic kinase activity that explains the previously reported ability of ILK to phosphorylate b1 integrin tail, PKB and GSK-3a in vitro (Hannigan et al., 1996; Delcommenne et al., 1998), this kinase activity is unlikely to be sucient to account for the rapid phosphorylation of S473 of PKB that is noted in vivo (Cohen et al., 1997; van Weeren et al., 1998). (3) Substitution of an FSF motif in ILK by either FDF or FEF restores activity to a kinase-dead ILK protein (DN-ILK) arguing strongly against direct phosphor- ylation of PKB by ILK (Figure 4). If ILK isn't responsible for the direct phosphoryla- tion of S473 of PKB then this raises the questions of how does ILK function and what is the identity of PDK2? Two related models can be proposed for ILK function. In both models, ILK initially binds ATP in its ATP binding pocket and is then able to become Figure 4 E€ect of mutations in the FSF motif of integrin-linked phosphorylated on the FSF motif. If this phosphoryla- kinase on phosphorylation of protein kinase B in serum starved tion is mediated by a low intrinsic kinase activity this transiently transfected COS cells. COS cells were transiently then explains the ability of DN-ILK to act as a transfected with wild-type ILK (lane 1) or the ILK mutant dominant negative inhibitor, since autophosphoryla- plasmids, DN-ILK (lane 2), ILK-FDF (lane 3), ILK-FEF (lane 4), ILK-FAF (lane 5), DN-ILK-FDF (lane 6), DN-ILK-FEF tion on the FSF motif will be blocked in this instance. (lane 7), DN-ILK-FAF (lane 8), or left untransfected (lane 9). 3-phosphoinositides regulate access of ATP into the RIPA bu€er extracts were fractionated by SDS ± PAGE, blotted ILK ATP binding pocket in an analogous way to that to PVDF membrane and probed with antisera speci®c for; PKB, proposed for the regulation of ATP access into the phospho-Ser 473 PKB, phospho thr-308 PKB or ILK. Where ATP binding site by phosphorylation in the atrial indicated, cells were treated with 100 nM wortmannin for 30 min prior to cell lysis. Equal amounts of protein were loaded per track natriuretic receptor (Foster and Garbers, 1998; Potter Regulation of protein kinase B by ILK DK Lynch et al 8030 and Hunter, 1998). Indeed the glycine rich loop of the phosphoserine 136 BAD (New England Biolabs); GSK-3 (a ILK ATP binding pocket overlaps with sequences and b), phosphoserine 21 GSK-3a (Upstate Biotechnologies predicted by Delcommenne et al., 1998 to be Inc.); phospho-Map kinase, Map kinase (Promega). ILK responsible for the binding of 3-phosphoinosides by antiserum was raised against a TrpE-ILK fusion protein ILK. Subsequently, ILK phosphorylated on the FSF encompassing the N-terminal ankyrin repeat region of ILK (residues 1 ± 171). motif either (a) activates PDK2 activity or (b) inhibits a S473 phosphatase activity. Both scenarios result in increased phosphorylation of S473 of PKB. Although this model predicts the ILK-FDF and ILK-FEF Clones mutants no-longer require 3-phosphoinositides for Expression constructs were made by subcloning cDNAs for function, wortmannin does partially inhibit the ability ErbB4 CYT-1 and CYT-2 and ILK from pBluescript into the of these mutants to induce phosphorylation on S473 of expression vector pMT2 (Sambrook et al., 1987) with PKB (Figure 4). We suspect this is because 3- modi®ed cloning sites including NotI. DN-ILK was phosphoinositides are still required for proper mem- constructed by PCR mutagenesis. Oligonucleotides including restriction sites for BsrD1 5'-CACAGAATTCGCAATGA- brane targeting and unfolding of PKB. Current CATCGTTGTGATGGTGCTGAAGGTTCGAG (5 prime) experiments are aimed at distinguishing between these and NdeI5'-TAGAGGGATCCATATGGCATCCAG (3 two possible methods of action of ILK. Model A prime) were used to amplify a 188 bp fragment containing predicts that PDK2 activity lies in a wortmannin the desired lysine to methionine change (residue 220 ± in insensitive kinase that associates with ILK phosphory- bold). This was cloned into pBluescript as a EcoRI-BamHI lated on the FSF motif. However, this model is still fragment and the change con®rmed by DNA sequencing. A consistent with PDK2 activity being 3-phosphoinosi- BsrDI-NdeI fragment (a silent G-A polymorphism creates a tide dependent in vivo, since there is a requirement for NdeI site in our ILK cDNA) was re-cloned into pBluescript- 3-phosphoinositides for the initial phosphorylation of ILK and the ILK cDNA with the lysine to methionine ILK on the FSF motif. In vitro, PDK1 gains PDK2 mutation subcloned into pMT2 as a NotI fragment. Mutation of the FSF motif was performed by PCR activity when complexed with PRK2 or c-terminal directed mutagenesis. PCR primers were constructed covering peptides from PRK2 containing an FEF sequence and including the NdeI(5'-CTCTAAGCTTCACACACTG- motif (Balendran et al., 1999). However, it is unlikely GATGCCATATGGAT) and BstXI sites. Primers covering that the phosphorylated FSF motif of ILK functions in the BstXI site included the following amino-acid substitutions a similar manner, as, unlike the PRK2/PDK1 complex, FSF to FEF: (5'-TGTGTCTAGACCAGGACATTG- which retains kinase activity towards T308, we see no GAATTCGAACTTGACATCAG); FSF to FDF (5'- increase in T308 phosphorylation when ILK is TGTGAATTCCAGGACATTGGAAATCGAACTTGACA- expressed and phosphorylation on S473 of PKB is TCAG); FSF to FAF (5'-TGTGAATTCCAGGACATTG- increased. In addition, we are unable to detect PDK1 GAACGCGAACTTGACATCAG). Fragments were ampli- in ILK-containing immuneprecipitates (data not ®ed by PCR using Pfu DNA polymerase (Stratagene) and subcloned into pMT2-ILK or pMT2 DN-ILK as NdeI-BstXI shown). Model B, where ILK inhibits a S473- fragments. Insert segments of all vectors were con®rmed by phosphatase activity may be more likely given the DNA sequence analysis. large increase in S473 phosphorylation promoted by ILK expression. Whilst serum-starvation of COS cells only decreases PKB activity twofold (Meier et al., COS Transfections 1997), treatment with phosphatase inhibitors increases activity several-fold (Andjelkovic et al., 1996; Meier et COS1 cells were grown in Glasgow's MEM (Life Technol- al., 1997; Millward et al., 1999). ogies) supplemented with 10% v/v newborn calf serum and In this paper, we show that full activation of PKB in 2mM L-glutamine. Transfections were performed with Fugene 6 (Boehringer) and cells left for 72 h before harvest. a model COS cell system requires both the recruitment For analysis of BAD, pEBG-mBAD (New England Biolabs), and activation of PI3-kinase by ErbB4 and ILK. Since encoding a 49KD murine-BAD fusion protein, was ILK is regulated by integrins (Hannigan et al., 1996), transiently co-transformed with other plasmids. Cells were PKB activation is therefore dependent on integrin- and serum starved for 16 h, washed twice in ice-cold phosphate growth factor-mediated signals. Dual regulation of bu€ered saline (PBS) and lysed for 15 min on ice in PKB activity in this way may be required to drive cell- radioimmune precipitation assay (RIPA) bu€er 0.5% w/v cycle progression, and/or cell survival, both biological sodium deoxycholate, 150 mM sodium chloride, 1% v/v responses known to require joint input from growth NP40, 50 mM Tris-HCL pH 8.0, 0.1% w/v SDS, 10% v/v factor- and extracellular matrix-mediated signals. The glycerol, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM 71 71 mechanism by which ILK regulates S473 phosphoryla- PMSF, 10 mg ml aprotinin, 10 mg ml leupeptin. Insoluble material was removed by centrifugation (12 000 g tion on PKB is unclear, although our results strongly for 10 min at 48C). suggest it is unlikely to be due to direct phosphoryla- tion of PKB by ILK. Rather, our results suggest that PDK2 activity residues in a distinct molecule and ILK Western blots functions as an adaptor protein that either activates PDK2 or inhibits a S473 phosphatase. Cells were lysed in 200 ml RIPA lysis bu€er and protein content estimated using Bicinchoninic acid Protein Assay Reagent (Pierce). 20 mg protein was boiled in SDS sample Materials and methods bu€er and resolved on a 7.5% SDS-polyacrylamide gel. Proteins were transferred to Immobilon PVDF membrane Antibodies (Millipore) by electro-blotting and probed with appropriate antibodies at the recommended dilutions. Protein detection ErbB4, Santa Cruz C-18; phosphoserine 473 Akt (PKB), was performed using ECL (Amersham Life Science) followed phosphothreonine 308 Akt (PKB), Akt (PKB), BAD, by autoradiography. Regulation of protein kinase B by ILK DK Lynch et al 8031 C2-ceramide-induced apoptosis ILK sequences This was performed as described in Zhou et al. (1998). Partial C. elegans sequences with homology to human ILK were identi®ed in EST databases (GenBank). PCR primers were constructed and RT ± PCR carried out using mRNA Detection of apoptosis from whole embryos to obtain full-length cDNAs. Screening TUNEL staining was performed using the Apoptosis of EST databases (GenBank) identi®ed EST clones covering Detection System, Fluorescein (Promega) Flow-cytometry/ the N-terminus of ILK from D. melanogaster. Two of these FACS analysis with propidium iodide. Cells (106) were (LD02317 and LD08711: Berkeley Drosophila Genome harvested by mild trypsinisation and pelleted (1500 r.p.m., Project) were obtained from Research Genetics Inc and 5 min). Cell pellets were permeabilized with 1 ml 3.7% sequenced. DNA sequence analysis was performed using paraformaldehyde (5 min 228C), pelleted, washed with PBS Perkin-Elmer ABI Prism 377 Sequencers and BigDye and resuspended in 1 ml 0.1% Triton X-100, 1% BSA in Terminator Chemistry (Perkin-Elmer, Foster City, CA., PBS, incubated for 5 min at 48C, repelleted (3000 r.p.m., USA). 5 min) and resuspended in 1 ml PBS containing 5 mgml71 Accession numbers for sequences are C. elegans propidium iodide and 100 mgml71 ribonuclease. Cells were (AJ249344) and D. melanogaster (AJ249345). incubated for 1 h at 228C in the dark and cell ¯uorescence measured at 620 nm (excitation 488 nm) using a FACscan ¯ow cytometer and CELLQuest acquisition and analysis software (Becton Dickinson). Acknowledgements Trypan Blue exclusion assay. Cells were harvested by mild We thank Simon Barry, Harren Jhoti, Rob Harris and trypsinization, pelleted (1500 r.p.m., 5 min) and resuspended Peter Soden for helpful discussions and technical advice. in 1 ml growth medium. 50 ml cells was added to 50 ml 0.4% This work was supported by a MRC collaborative Trypan Blue in PBS with mixing. After 2 min, stained and studentship award between GlaxoWellcome and Cam- unstained cells were quantitated by counting with a bridge University (DK Lynch) and a grant from the haemocytometer. A minimum of 600 cells were counted per Association for International Cancer Research (PAW point. Edwards).

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