LIGAND-INDEPENDENT ACTIVATION OF THE PLATELET-DERIVED GROWTH-FACTOR-BETA RECEPTOR - REQUIREMENTS FOR BOVINE PAPILLOMAVIRUS E5-INDUCED MITOGENIC SIGNALING

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a fun ctional assay to test the ability of PDGF beta receptor mutants to med iate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 ce lls are strictly dependent on interleukin-3 for growth, but coexpressi on of the wild-type PDGF beta receptor and the E5 or v-sis-encoded pro tein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 p rotein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor . The tyrosine kinase activity of the receptor was necessary for E5-in duced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-bind ing domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic acti vity, demonstrating that E5-mediated receptor activation is ligand ind ependent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested tha t the wild-type PDGF beta receptor can generate multiple independent m itogenic signals. Importantly, these mutants dissociated two activitie s of the PDGF beta receptor tyrosine kinase, both of which are require d for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and ( ii) phosphorylation of substrates other than the receptor itself.