Transforming growth factor-β-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs

Abstract

The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCι/λ and PKCζ serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCι/λ as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCι/λ. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential.

Fig. 1

PKCs ζ and ι interact with the KLF family zinc finger domain in vitro and in HeLa cells. a Schematic representation of the domain structure of TIEG1. The three zinc finger motifs constituting the DNA-binding domain are represented by filled boxes. The TIEG1 region isolated in the yeast two-hybrid screen is indicated by the solid line below the schematic. b Whole-cell extracts from HeLa cells were incubated with equal amounts of bacterially expressed GST or GST-TIEG1 immobilized on glutathione-sepharose beads. The presence of aPKCs in the pulled-down proteins were examined by immunoblotting using αPKCζ(C20) antibody that recognizes both PKCζ and PKCι. c U2OS cells were transfected with either GFP or GFP-TIEG1. The cells were lysed 24 h post transfection, and immunoprecipitations were performed using anti-GFP antibody. Western blots of the immunoprecipitates and of the cell extract were revealed using monoclonal anti-PKCλ antibody (left panel) and immunoprecipitated GFP or GFP-TIEG1 proteins were visualized using polyclonal anti-GFP antibody (right panel). d Myc-tagged PKCλ was in vitro translated in the presence of [³⁵S] methionine and incubated with equal amounts of either glutathione-sepharose beads coupled GST or GST-tagged KLF family proteins. The pulled-down proteins together with 5% of the input were subjected to detection by autoradiography (upper panel). The levels of GST or GST-tagged proteins used in the GST pulldown assays were examined by Coomassie brilliant blue staining (lower panel). The data shown in b, c, and d correspond to a representative experiment out of two performed

Fig. 2

The KLF family proteins are phosphorylated by the atypical PKCs ζ and ι. a, c Both PKCζ and PKCι phosphorylate TIEG1 (a) and KLF4 (c) in vitro. The phosphorylation assays were performed for 20 min at 30°C in 30-μl reaction volume containing MBP, and MBP-TIEG1 or MBP-KLF4 as substrate, 50 ng recombinant active PKCζ (upper panels) or PKCι (lower panels) 60 μM unlabeled ATP and 2 μCi [³²P]-ATP. The reactions were analyzed by autoradiography. b, d The phosphorylation of TIEG1 (b) and KLF4 (d) by PKCζ is linear with time. The upper panels are bar graphs showing the quantification of the phosphorylation levels of TIEG1 and KLF4 over the indicated time points, respectively, and the lower panels are the corresponding autoradiographs. e, f The DNA-binding domains of KLF family proteins are targets for PKCζ-mediated phosphorylation. GST, GST-TIEG1(1–362) and GST-TIEG1(DBD) (e), and GST, GST-tagged DBDs of KLF family proteins (f) were subjected to in vitro phosphorylation and subsequent analysis by autoradiography. The data in a, c, e and f are representative of three independent experiments. The data in b and f are shown as the mean (± SD) of four independent experiments

Fig. 3

PKCζ phosphorylates TIEG1 at Ser 384 located in zinc finger 1 and Thr 445 located in zinc finger 3. a Amino acid sequence alignment of the DBDs of the KLF family proteins. Conserved residues are shaded, and the core zinc finger domains are indicated by thick lines and labeled ZF 1–3. The locations of the phospho-acceptors Ser 384 and Thr 445 residues are boxed. Below and to the left is the structure of the zinc finger protein ZNF268 [57] with the location of the phosphorylation sites in the recognition helixes of zinc fingers 1 and 3 indicated by arrows. Below and to the right are the amino acid sequences of zinc fingers 1 and 3 in TIEG1. The amino acids within the recognition helix that are proposed to contact the DNA bases are indicated by solid lines. The prediction is based on the NMR structure of Sp1 [58]. Ser 384 and Thr 445 are shown in bold. b, c Mutations of TIEG1S384 and TIEG1T445 (b), and Sp1S641 and Sp1S702 (c) to alanine significantly reduce their PKCζ-mediated phosphorylation. Equal amounts of GST and GST-tagged TIEG1 proteins (b), GST and GST-tagged Sp1 proteins (c) were subjected to in vitro phosphorylation assays; detection and quantification as described above. Shown are the quantified phosphorylation levels as bar graphs (upper panels), autoradiographs (middle panels) and Coomassie staining indicating the levels of GST and GST-tagged proteins used in the in vitro kinase assays (lower panels). Each bar in b and c represents the mean (± SD) of four independent experiments

Fig. 4

PKCζ-mediated phosphorylation of TIEG1S384 impairs its DNA-binding activity. a–c Gel mobility shift assays (GMSA) demonstrating the effects of phospho-mimicking substitutions or in vitro phosphorylation on the DNA-binding ability of KLF proteins. a Substitution of Ser 384 to glutamate abolishes the DNA-binding ability of TIEG1. GMSA using GST and GST-fused TIEG1 proteins, and [³²P]-ATP labeled oligonucleotides containing consensus TIEG1-binding sites described in [17]. The binding reactions were separated on non-denaturating polyacrylamide gels that were vacuum dried and analyzed by autoradiography (upper panel). The lower panel shows control protein staining indicating the amounts of proteins used in the assays. b Pre-phosphorylation of TIEG1(DBD) by PKCζ also impairs DNA binding. Here, GST and GST-fused TIEG1 proteins were pre-incubated with PKCζ in the presence or absence of ATP prior to GMSA. c PKCζ-mediated phosphorylation of the DBDs of KLF4 and Sp1 does not impair their DNA-binding activity. GMSA with GST and GST-KLF4(DBD) (left panel), and GST and GST-tagged Sp1proteins (right panel). The proteins were pre-treated with PKCζ and/or ATP prior to GMSA using [³²P]-ATP labeled oligonucleotide containing consensus KLF-binding sites described in [18]. The data shown are representative of three independent experiments

Fig. 5

Mutation of TIEG1 Ser 384 and Thr 445 to glutamate induces subnuclear relocalization. a The phospho-mimicking mutants of TIEG1 are enriched in round nuclear bodies. HeLa and/or U2OS cells were transfected with an expression construct for EGFP-TIEG1 or EGFP-tagged TIEG1 mutants and imaged by confocal laser fluorescence microscopy 24 h post transfection. b The TIEG1 mutants display mobility similar to the wild-type TIEG1. HeLa cells were transfected with the indicated constructs, and 24 h post transfection the cells were imaged and fluorescence signals were collected before and after photobleaching a portion of the nucleus. The recovery kinetics were calculated after double normalization of the initial fluorescence, corrected for background and loss of fluorescence caused by imaging. The graph presented here shows the average intensity of the bleach spot of 9–10 independent cells for the indicated time points. Error bars represent the SD values between various experiments

Fig. 6

The TIEG1T445E mutant displays enhanced transactivation potential. a U2OS cells were transiently transfected with 0.1 μg of the reporter p21-LUC, together with the expression vectors for TIEG1 and TIEG1 mutants as indicated and subjected to reporter gene assay analysis. The data represent the mean of two independent experiments performed in triplicates. Error bars represent the SD values between various experiments. b Total cell extracts from the transfected U2OS cells were prepared, and the levels of TIEG1 proteins were analyzed by Western blotting using anti-GFP antibody (upper panel) and then with anti-actin antibody as loading control (lower panel). c All KLF family proteins tested except KLF4 are potential activators of the p21^Cip1/WAF1 promoter. The reporter gene assay data shown is from one experiment performed in triplicate, which is representative of three independent experiments each performed in triplicate. Error bars represent the SD values between the parallels. d Western blot analysis of extracts from transfected U2OS cells to show the expression level of the various KLF family proteins

Fig. 7

KLF family proteins have the ability to homo- and heterodimerize via their DNA-binding domain. a–c GST pulldown assays were carried out using bacterially expressed GST, or the indicated GST-tagged proteins, and in vitro translated either full-length HA-TIEG1 (a, b) or GFP-TIEG1(DBD) (c). Autoradiographs are presented in the upper panels, whereas Coomassie brilliant blue stained SDS-polyacrylamide gels are shown in the lower panels. c, d TIEG1 homodimerization is not mediated by nucleic acids in the reaction mixture. GFP-TIEG1(DBD) was in vitro translated and divided into two. One half was used directly for GST pulldown assays (c, right panel), and the remaining half was treated with 250 units of benzonase for 20 min at room temperature prior to GST pulldown assays (c, left panel). d The benzonase used in the assay was active. The expression construct, GFP-TIEG1(DBD), was either treated with benzonase or left untreated, and the reaction analyzed on a 0.7% agarose gel followed by ethdium bromide staining. Data shown are representative of two independent experiments