Specific association of Mil/Raf proteins with a 34 kDa phosphoprotein.

Mil/Raf protein kinases are intermediates in signaling pathways leading to differentiation, mitogenesis and cellular transformation. To gain insight into the activity of Mil/Raf kinases at the molecular level we aimed to identify proteins specifically interacting with Mil/Raf proteins. A phosphoprotein of 34 kDa (pp34) was found to be associated with c-Raf as well as with viral and activated forms of Mil/Raf proteins in exponentially growing interphase cells. pp34 association was not detectable in mitotic cells. Serum stimulation or coexpression of activated Ras led to decreased electrophoretic mobility of pp34 complexed to Mil/Raf proteins while serum starvation rendered pp34 undetectable. Moreover, the association with pp34 became undetectable in parallel with the onset of morphological cellular transformation caused by overexpression of a constitutively activated mutant of c-Raf in an inducible expression system. Thus, the association of Mil/Raf proteins with pp34 is altered in the course of cell cycle progression, serum stimulation and cellular transformation. These events represent hallmarks of cellular Mil/Raf functions, rendering pp34 a candidate protein involved in Mil/Raf function