Fig. 1. ILKAP structure and alignment with PP2C. (A) Deduced protein sequence of ILKAP cDNA. Sequence was translated from the H4C11-30 cDNA sequence using Gene Inspector v. 1.5 software (Textco, Inc., New Hampshire). PP2C boxes 1 and 2 are underlined, and bold letters indicate the DGH active site triplet. (B) Amino acid residues 115–392 of ILKAP were aligned using the Clustal V algorithm (Gene Inspector 1.5) and the align ment edited to highlight identities (black) and conserved (shaded) amino acid residues, using Genedoc v. 2.6.01 (www.psc.edu/biomed/genedoc/).

Fig. 6. ILKAP selectively inhibits phosphorylation of GSK3β at Ser9. (A) ILKAP clone wt/6-4 and vector control transfectant cells were serum starved and pre-treated or left untreated, prior to plating on FN. Control cells showed FN-dependent induction of Ser9 phosphorylation, which was inhibited in the wt/6-4 cells induced with muristerone A. Parallel blots were probed with phosphospecific antibody for PKB Ser473. Induction of ILKAP had no effect on Ser473 phosphorylation. Blots were stripped and reprobed with appropriate antibodies, for total ILK1 and GSK3 protein levels. (B) ILKAP clone wt/6-10 was treated as in (A), in parallel with the non-clonal transfectant cell line expressing a catalytically inactive H154D ILKAP mutant. Muristerone induction of ILKAP suppressed FN-induced Ser9 phosphorylation; however, the catalytic mutant did not inhibit this phosphorylation. Parallel blots were probed for changes in Ser 21 of GSK3α/β, which was not detectably affected by ILKAP activity. Blots were stripped and reprobed for total GSKβ protein levels.

Fig. 3. PP2C activity of recombinant ILKAP expressed in mammalian cells. (A) HEK 293 cells stably expressing the synthetic ecdysone receptor (pVgRXR) were transiently transfected with pIND/V5-ILKAP, in order to characterize muristerone A-induced phosphatase activity. Vector control cells were the stable pVgRXR 293 transfectants. Muristerone A treatment of the pIND/V5-ILKAP cells induced OA- and EGTA-resistant PP2C ∼3-fold relative to uninduced pIND/V5-ILKAP transfected cells; however, it did not raise PP2C activity of vector control cells above background. Muristerone-induced S/T phosphatase activity was partially sensitive to inhibition by Mg²⁺, as reported for rat PP2C (Tong et al., 1998). (B) pVgRXR-expressing 293 cells were transiently transfected with pIND/V5-ILKAP plasmids encoding wild-type ILKAP, or the indicated ILKAP catalytic mutants. Muristerone-induced PP2C activity from the wild-type ILKAP transfectants was ∼5-fold above background levels seen with the mutant ILKAP transfectants. All assays were performed in triplicate, with error bars indicating 1 SEM. Control experiments confirmed that this phosphatase activity was dependent on Mn²⁺, and not inhibited by OA or EGTA, as expected of PP2C enzymes. Thus, all assays in (A) and (B) were carried out in the presence of Mn²⁺, OA and EGTA.

Fig. 5. ILKAP selectively inhibits ILK1 kinase activity. (A) Cytoplasmic lysates from ILKAP clone wt/6-10 were analysed in ILK1 (top panel) or Raf-1 (middle panel) immune complex kinase assays using MBP as exogenous substrate. Cells were pre-treated for 36 h with or without muristerone A, detached, and replated for 20 min on BSA or FN in 10% serum, or treated for 10 min with 100 ng/ml IGF-1, to induce high levels of ILK1 activity. Lysates were controlled for equal levels of p59^ILK1 protein (bottom panel). (B) The stable ILKAP clones wt/6-4 and wt/6-10, and a non-clonal H154D pooled transfectant cell line, were treated with muristerone A for 36 h to induce recombinant protein expression. Subsequently, these cells were plated on FN or poly-l-lysine (PLL) for 10 min, after which cell lysates were assayed for ILK1 activity by immune complex kinase assays (MBP substrate), and also for PKB Ser473 phoshorylation by western blotting with a pSer473-specific antibody. Induction of ILKAP, but not the H154D mutant, inhibited ILK1 immune complex kinase activity. Ser473 phosphorylation was not inhibited in parallel with ILK1 inhibition. Plating on PLL did not induce ILK1 activity or Ser473 phosphorylation.

Fig. 2. ILKAP transcript is preferentially expressed in skeletal muscle. (A) A partial cDNA representing amino acid residues 269–392 of ILKAP was ³²P labelled, and used to probe a northern blot of poly(A)⁺ RNAs from human striated and smooth muscle tissues. Sk, skeletal muscle; U, uterus; C, colon; Sm, small intestine; B, bladder; H, heart; St, stomach; P, prostate. (B) Left panel: a polyclonal antibody was raised to an ILKAP fusion protein (residues 269–392) and affinity purified over an immobilized ILKAP column. Pre-immune serum from the same rabbit was subjected to the same purification protocol, as a negative control. Cytoplasmic lysates of HEK 293 cells were analysed by western blotting using the ILKAP immune and pre-immune sera as indicated. Right panel: cytoplasmic 293 cell lysates were immunoprecipitated with rabbit IgG, or affinity-purified rabbit polyclonal ILK1 antibody, covalently coupled to CNBr-activated Sepharose beads. Unfractionated cytoplasmic lysates and ILK1 immunoprecipitates each indicated a single ILKAP band, migrating with an apparent mol. wt of 47 kDa. Immune complexes were analysed by western blotting, using affinity-purified ILKAP antibody. Arrowheads indicate p47^ILKAP migration on 12% SDS–PAGE.

Fig. 4. ILKAP and ILK1 associate in mammalian cells. (A) HEK 293 were transiently co-transfected with pIND/V5-ILKAP and pVgRXR plasmids. After 24 h of culture, transfected cells were then induced with 1 or 2 µM muristerone A, or left untreated, for the indicated periods. Cytoplasmic lysates were analysed by western blotting for expression of the V5 epitope, migrating at the appropriate size of ∼50 kDa. (B) 293 cells were transiently co-transfected, as above. After 24 h, transfectants were induced with muristerone A for the indicated times. Cytoplasmic lysates of these cells were immunoprecipitated with affinity-purified ILK1 antibodies, and the resulting immune complexes analysed by SDS–PAGE and western blotting for the presence of the V5-tagged ILKAP protein. (C) 293/pVgRXR and wt/6-10 clones were cultured under normal conditions, prior to induction with 1 μM muristerone A for 36 h. Cytoplasmic lysates (WCE; top panel) were immunoprecipitated (bottom panel) with anti-Raf-1 antibody (C-20; Santa Cruz). Immune complexes were run on 10% SDS–PAGE and transferred to PVDF membranes. Membranes were blotted with anti-V5 monoclonal antibody, and visualization was by ECL. (D) 293/pVgRXR cells were transiently transfected as in (A), with the indicated ILKAP mutants. Transfectant cultures were lysed and subjected to immunoprecipitation with affinity-purified ILK1 antibodies. Complexes were analysed for co-precipitating V5-tagged ILKAP and mutants, by SDS–PAGE and western blotting with anti-V5 monoclonal antibody. ILKAP^V indicates variant ILKAP recombinant proteins.

Fig. 7. Tcf/Lef factor activation is selectively inhibited by ILKAP. (A) TOPFlash and FOPFlash (mutant Tcf site) reporter plasmids were co-transfected with pRSV/βgal into 293/pVgRXR vector control, ILKAP wt/6-4 and wt/6-10, H154D and PP2Cα expressing lines (dark bars). Cells were untreated or muristerone A was added for 36 h to induce recombinant protein expression, and resulting Tcf-driven or ISRE-luc-driven luciferase reporter gene activity assayed. In the case of the ISRE-luc transfectants, recombinant human IFN-α was added (500 IU/ml) for the last 18 h of the induction period. Control experiments confirmed ∼3-fold induction of ISRE-luc activity by this IFN treatment (data not shown). Luciferase activity is presented as the muristerone-induced/uninduced ratio, also corrected for luciferase activity from duplicate FOPFlash mutant reporter transfections in the case of FOPFlash results. (B) The Tcf-luciferase reporter construct TCF-pGL2 (see Materials and methods) was co-transfected with pRSV/βgal into the pVgRXR/293 vector control and ILKAP clone wt/6-10 cell lines. Transfectants cultured in 10% FCS (a), 10% FCS + 50 ng/ml IGF-1 (b) or 10% FCS + 100 nM insulin (c) were not induced or induced with muristerone A, and subsequently assayed for TCF-driven luciferase activity. Each transfection in (a), (b) and (c) was controlled internally and normalized to β-galactosidase activity, to account for transfection efficiencies.