Adaptor Protein Lnk Binds to PDGFRA, PDGFRB and FIP1L1-PDGFRA, but Not to the TEL-PDGFRB Fusion Protein.

PDGFRA and PDGFRB (platelet derived growth factor receptors alpha and beta) are frequently expressed on malignant hematopoietic cells and regulate various cellular responses such as development, proliferation, differentiation, cell survival and cellular transformation. Stimulation by either autocrine loops or constitutional activation by chromosomal translocation (i.e. chronic myelomonocytic leukemia [CMML, TEL-PDGFRB] or chronic eosinophilic leukemia [CEL, FIP1L1-PDGFRA]) makes them important factors in development of hematopoietic disorders. Normally, interaction with the ligand PDGF, induces dimerization of two distinct receptor subunits, resulting in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. We hypothesized that one such protein may be the adaptor Lnk, a negative regulator of several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain, a pleckstrin homology domain (PH) and a dimerization domain (DD). The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. We investigated the ability of Lnk to bind to PDGFRA, PDGFRB, FIP1L1-PDGFRA and TEL-PDGFRB. To determine the domain of Lnk that is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including:

1. a mutation in the SH2 domain (R392E);

2. deletion of the SH2 domain;

3. deletion of the PH and SH2 domains and

4. a construct only containing the DD domain.

293T cells were co-transfected with cDNAs encoding either PDGFRA, PDGFRB or one of the translocation products and either wild-type or mutant Lnk. Whole cell lysates were used to perform immunoprecipitation with either V5-tag or PDGFR antibodies. Binding of Lnk and PDGFR was detected by Western blot probed with PDGFR or V5-tag antibodies. NIH3T3 cells were transfected either with empty vector or Lnk cDNA, transfectants were selected for 5 days with G418, serum starved for 16 hours and induced with PDGF for 10 minutes. Phosphorylation of downstream targets of PDGFRA and PDGFRB was detected by Western blot. Our data showed that Lnk bound to PDGFRA and PDGFRB only after exposure of the cells to PDGF and to the FIP1L1-PDGFRA fusion protein independent of PDGF exposure. Mutation or deletion of the Lnk SH2 domain abolished binding completely in PDGFRA and FIP1L1-PDGFRA, but just partly in PDGFRB. Expression of Lnk in NIH3T3 cells inhibited phosphorylation of ERK after treatment with PDGF. In other experiments, we determined that Lnk bound the juxtamembrane region of this class of receptors. Interestingly, the TEL-PDGFRB fusion protein was unable to bind Lnk, although its breakpoint in PDGFRB is distal to the juxtamembrane domain and the whole intracellular region of PDGFRB is included in the fusion protein. Further exploration of the mechanisms by which Lnk affects wild-type or PDGFR fusion product will provide insight into the molecular pathophysiology of myeloid disorders and could help develop new treatments.