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Novel interaction partners of the TPR/MET tyrosine kinase

Institute of Human Genetics, University Clinics of Heidelberg, Heidelberg, Germany

¹Correspondence: E-mail: hans_janssen{at}med.uni-heidelberg.de

SPECIFIC AIM

An intriguing issue in the field of tyrosine kinase receptors is how activation of different growth factor receptors can elicit different cellular responses despite activating a common set of signaling molecules. Qualitative and quantitative differences have been proposed to explain this phenomenon. The MET tyrosine kinase receptor was discovered as an oncogene and its ligand identified as a mitogenic factor for hepatocytes (hepatocyte growth factor, HGF) and a potent motility factor (scatter factor, SF), referred to as HGF/SF. It has been established that MET plays a pivotal role in development and human cancer as MET controls cell migration, growth, invasion, and metastasis in cancer cells. To gain further insight into how a large number of highly different cellular responses can be transmitted through activation of the MET receptor, we set out to investigate whether novel interaction partners of TPR/MET could be identified by application of the yeast two-hybrid assay.

PRINCIPAL FINDINGS

1. Identification of novel interaction partners of the MET tyrosine kinase receptor
To screen for novel putative interaction partners of MET, we used the oncogenic TPR/MET fusion gene as a bait and a rat embryo cDNA library as a prey in a yeast two-hybrid screen. TPR sequences encode a leucine zipper dimerization motif, leading to constitutive dimerization and thus tyrosine phosphorylation of the TPR/MET fusion protein. Of 290 His-positive clones, 86 were found to be positive in the qualitative ß-galactosidase assay for LacZ reporter expression, 31 showed weak and 55 showed strong ß-galactosidase activity. Twenty-five of the 55 yeast colonies tested were positive for GRB-2 sequences by hybridization analysis. Retransformation of all 30 GRB-2-negative His-positive transformants with a bait plasmid containing the complete tpr/met construct, the met part (cytoplasmic portion of MET), or the tpr part, respectively, showed moderate to high interaction with TPR/MET and low to high interaction with the cytoplasmic part of the MET receptor alone. One of 30 clones showed interaction with the TPR portion of TPR/MET. Sequence analysis revealed 13 clones that are suspected to be false positives and were not investigated further (e.g., mitochondrial cytochrome oxidase, ribosomal protein B20). Putative physiological interaction partners of the MET receptor tyrosine kinase, as identified by our yeast two-hybrid analyses, are shown in Table 1 . mTID-1 was isolated from five independent clones; DAPK-3, CENPC, and DCOHM could be isolated twice and four interaction partners were detected only once in our screen (SNAPIN, VAV-1, SNX-2, and SMC-1, respectively).

2. Verification of interaction between TPR/MET and identified partners by mammalian two-hybrid analysis
To verify that interactions between TPR/MET and new putative substrates discovered in yeast can take place in higher eukaryotic organisms, we performed a mammalian two-hybrid assay. To learn more about binding affinities of putative substrates to MET and the oncogenic fusion protein TPR/MET, we performed mammalian two-hybrid studies with both constructs serving as "bait." Activation of the MET-part alone is ensured through dimerization of the GAL4 DNA binding domain of the bait vector. HEK-293 cells were cotransfected with tpr/met- and met constructs subcloned into pBind, the various interaction partners subcloned into pACT vector and the reporter vector pG5luc (reporter gene: firefly luciferase). Firefly luciferase activity of the reporter was significantly higher with all interaction partners than the empty bait vector used as a control (set at 100%). Binding to MET is stronger than to TPR/MET for most interaction partners except for SMC-1, which shows stronger affinity to TPR/MET than to MET. GRB-2, a well-known strong interaction partner of MET, served as positive control (Table 1) .

3. Far-Western analysis supports direct interaction between TPR/MET and its partners
To confirm our results from two-hybrid assays, we examined interactions by far-Western analysis. Insert DNAs of interaction partners were cut out of pGADGH plasmid and recloned in frame into a GST fusion protein vector. Bacterial lysates of IPTG-induced and noninduced GST-proteins were separated on gels and analyzed by Western blot. Filters were incubated with anti-GST antibodies or incubated with purified and [³²P]-labeled HA-tagged TPR/MET protein, respectively. The oncogenic variant of the MET receptor was used to ensure constitutive activation of the receptor tyrosine kinase. Successful induction of GST fusion proteins was demonstrated. Incubation of Western blots with labeled TPR/MET protein showed a strong interaction with DAPK-3, SMC-1 and GRB-2. Moderate binding was observed with CENPC and mTID-1 and a weak interaction was demonstrated with SNAPIN, DCOHM, VAV-1, and SNX-2 (Table 1) .

4. In vivo interaction between TPR/MET and its substrates
Coimmunoprecipitation analysis represents one of the most rigorous methods to demonstrate protein-protein interaction in vivo. Therefore, cDNA fragments of the various interaction partners were cloned into an eukaryotic expression vector exhibiting an Xpress, Myc, or Flag tag at the multiple cloning site’s NH₂ terminus. Tpr/met, tpr/met K241A and tprmet, were cloned into pMT2SM3xHA eukaryotic expression vector, containing a 3xHA tag. TPR/MET K241A is a kinase-negative variant of TPR/MET because of its inability to bind ATP. TPRMET contains the TPR portion solely. Coimmunoprecipitation studies were performed with extracts of transiently cotransfected HEK-293 cells. Total cell lysates were checked for proper expression of TPR/MET and the analyzed interaction partners were checked by immunoblot analysis with anti-HA, anti-Xpress, anti-Myc, or HRP-labeled anti-Flag antibodies, respectively (Fig. 1 ). Anti-HA antibodies were able to coimmunoprecipitate TPR/MET and the following interaction partners: DCOHM, VAV-1, SNX-2, and hTID-1. DCOHM, SNX-2, and hTID-1 were coimmunoprecipitated with the kinase inactive mutant TPR/MET K241A. It seems that these interaction partners can bind to TPR/MET independent of its phosphorylation status. SNAPIN, DAPK-3, and SMC-1 could be coimmunoprecipitated only with TPR/MET K241A and not with wild-type TPR/MET (Fig. 1) . None of the interaction partners were coimmunoprecipitated with TPRMET, implying that all partners bind to the MET portion of the TPR/MET tyrosine kinase and not to TPR. We were not able to demonstrate convincingly coimmunoprecipitation of CENPC. Coimmunoprecipitation of GRB-2 (the positive control) was positive with TPR/MET, but not with TPR/MET K241A, as expected.

View larger version (38K): [in this window] [in a new window]   Figure 1. Coimmunoprecipitation of putative interaction partners with TPR/MET, the kinase negative TPR/MET K241A and TPRMET. Total cell lysate was checked for proper expression of TPR/MET with anti-HA antibody (A) and for expression of the analyzed interaction partners with anti-Xpress, anti-myc (SNX2), or HRP-labeled anti-FLAG (Snapin) antibody (B). Corresponding to each interaction partner, column 1 shows cotransfection with pMT2SM-3xHA-tpr/met; column 2 with pMT2SM-3xHA-tpr/metK241A;and column 3 with pMT2SM-3xHA-tprmet. A) Mutated TPR/MET protein as a single band with a molecular mass of 64 kDa and the wild-type TPR/MET as one single band with a slightly larger size due to the fact that all TPR/MET proteins are phosphorylated on tyrosine. pMT2SM-3xHA-tprmet codes for a small portion of the TPR protein (that part of the TPR protein present in the TPR/MET fusion protein) with a molecular mass of 15 kDa. Coprecipitated proteins were detected using anti-Xpress (Invitrogen) or anti-myc antibodies (Babco) and HRP-conjugated secondary antibodies. Snapin was directly detected using HRP-labeled anti-FLAG antibody. Bands at 50 and 23 kDa resemble the heavy and light chain of the precipitating antibody. Coimmunoprecipitated proteins are marked by a black or white asterisk. hTID-1 and SNAPIN run at the same position as the heavy and light chains of immunoglobulin, respectively. However, compared with the control (TPR alone), signals of hTID-1 and SNAPIN in TPR/MET and/or TPR/MET K241A (C) are much stronger. For VAV-1 coimmunoprecipitation analysis no pMT2SM-3xHA-TPR/MET control is available. However coimmunoprecipitation analysis of VAV-1 with pMT2SM-3xHA-TPR/METK241A shows no interaction, implying that VAV-1 does not interact with the inactive/kinase-negative receptor as well as with the TPR portion alone. The position of migration and sizes (in kDa) of the protein molecular mass markers are indicated in the left borders.

CONCLUSIONS AND SIGNIFICANCE

Identification of several novel putative intracellular interaction partners of the TPR/MET tyrosine kinase strongly supports the idea that specific signaling molecules can be ascribed for individual aspects of the biological responses of the HGF receptor c-MET. Among these proteins is GRB-2, an interaction partner of MET, which confirms the suitability of our approach. Other known interaction partners of MET (e.g., PLC-, STAT-3, SHC) were not detected in our screen, which could be attributed to the fact that each cDNA library (we used a rat embryo library) has a limited capacity to detect interaction partners due to tissue distribution and sequence divergence between species.

Putative MET substrates detected by yeast two-hybrid became subject to an additional investigations clarifying different aspects of their putative binding to the RTK. Mammalian two-hybrid studies confirmed yeast data and proved that identified interactions are also recognized in an eukaryotic context. Far-Western analysis revealed that most of the interaction partners are able to bind TPR/MET directly in vitro. Finally coimmunoprecipitations provided strong evidence that SNAPIN, DCOHM, VAV-1, SNX-2, DAPK-3, SMC-1, and hTID-1 can interact with the TPR/MET tyrosine kinase in eukaryotic cells under most physiological conditions. Although far-Western analysis and coimmunoprecipitation were performed with TPR/MET and not with MET, all data suggest that these interaction partners would likely bind to the HGF receptor, c-MET. Interaction of CENPC with TPR/MET could not be proven directly by coimmunoprecipitation, perhaps due to incorrect folding or insolubility in the lysis buffer or insufficient expression of the respective protein. Coimmunoprecipitations provide us with additional information concerning the dependency of identified interactions on the phosphorylation status (i.e., the kinase activity of the TPR/MET protein).

The identification of several novel putative interaction partners of the TPR/MET tyrosine kinase shows that the intracellular signaling network associated with MET is by far more complex than previously supposed. Our data raise many questions that deserve further investigation. Functional studies facing these questions will contribute to a more integrative understanding of intracellular signaling after stimulation of the MET receptor.

View larger version (34K): [in this window] [in a new window]   Figure 2. Intracellular signaling of the MET receptor by the novel interaction partners. The novel MET signaling molecules (circles with gray background) and putative interaction partners (circles with white background) are depicted. Signaling molecules can interact with phosphorylated MET receptor (Yp), nonphosphorylated (line), or both (Y(p). Exact binding sites have not been assigned. Cellular processes regulated by the various proteins or protein complexes are indicated by arrows and boxes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1558fje;

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