Comparison of the 34,000-Da pp60src substrate and a 38,000-Da phosphoprotein identified by monoclonal antibodies.

M E Greenberg,G M Edelman

doi:10.1016/s0021-9258(20)82092-4

Abstract

One of the cellular targets of the pp60src tyrosine kinase is a phosphoprotein with a Mr = 34,000 and an isoelectric point of approximately 7.5 (Radke, K., Gilmore, T., and Martin, G. S. (1980) Cell 21, 821-828; Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836). We report here the preparation of monoclonal antibodies to partially purified 34-kDa protein and to a heretofore unrecognized phosphoprotein that is not a pp60src target. Two antibodies were initially obtained that recognized phosphoproteins in the Mr = 34,000-39,000 range. One of these antibodies immunoprecipitated a 34,000-Da protein which, on the basis of its molecular mass, phosphorylation state, and isoelectric point, was determined to be the 34-kDa pp60src substrate. The second monoclonal antibody bound to a 38,000-Da nucleolar associated protein, which appeared not to be a target of the pp60src kinase and was found by tryptic analysis to be structurally unrelated to the 34-kDa protein. The monoclonal antibody to the 34-kDa protein coupled to Sepharose CL-4B was used to purify the pp60src substrate to homogeneity in milligram quantities. Both the purified 34-kDa protein and the monoclonal antibody are currently being used in studies aimed at elucidating the structure and function of this pp60src target.

Highlights

One of the cellular targets of the pp60"" tyrosine cells has a molecular weight between 34,000 and 39,000 and a kinase is a phosphoprotein with aM, = 34,000 and an PI of7.5 [13,14,15]
We report here the preparation of monoclonal antibodies to partially purified 34-kDa protein and to a heretofore unrecognized phosphoprotein that is not a pp60""target
One of these antibodies immunoprecipitated a 34,000-Da protein which, on the basis of its molecular mass, phosphorylation state, and isoelectric point, was determined to be the 34-kDa pp60"" substrate.The second monoclonal antibody bound to a 38,000-Da nucleolar associated protein, which appeared not to be a targetof thepp60"" kinase and was found by tryptic analysis to be structurally unrelated to the 34-kDa protein

Summary

Using a rabbit antibody directed against the partialpluyrified

34-kDa protein, Erikson et al [16] observed that the phosphorylation of the34-kDaproteinattyrosine residues is significantly enhanced in cells infected by severaldifferent avian sarcoma viruses. Indirect experiments[17] suggested that the phosphorylated form of the 34-kDa protein is associated with the detergent-resistant cellular framework, and recent biochemical experiments [18]have IocaIized the 34-kDaprotein to the particulate fractionof cells. In an effort to characterize thispp60"" substrate in greater detail, we prepared monoclonal antibodies against a preparation of partially purified 34-kDaprotein.Two monoclonal pled to Sepharose CL-4B was used to purify the pp60"'" antibodies were obtained that immunoprecipitated different substrate to homogeneity in milligram quantities. Both phosphoproteinsinthe M , = 34,000-39,000 range. The transforming proteinosf a range of different avian sarcomaviruses have been observedto

MATERIAL SA N DM ETHODS

RESULTS

DISCUSSION

The specific antibodyhas provedusefulfor definingthe