Major substrate for growth factor-activated protein-tyrosine kinases is a low-abundance protein.

Abstract

A scarce, soluble, conserved protein was identified as the nonphosphorylated precursor of two related 42-kilodalton phosphoproteins that contain phosphotyrosine in mitogen-stimulated but not control fibroblasts.When cultures of resting fibroblasts are stimulated with many polypeptide or nonpeptide mitogens, there is an immediate increase in the phosphorylation of cell proteins at tyrosine (4,12,22). For example, the exposure of quiescent cultures of mouse 3T3 cells to epidermal growth factor or platelet-derived growth factor (PDGF) induces an approximate doubling of the protein phosphotyrosine content (4,12,22). An analysis of total cellular phosphoproteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that about 25% of the total increase in tyrosine phosphorylation occurs through a fivefold increase in the phosphotyrosine content of proteins in the 40-to 42-kilodalton range (22). Proteins in this size range are among those recognized by antibodies specific for phosphotyrosine in extracts of PDGF-treated cells (13,14).Two-dimensional polyacrylamide gel electrophoresis revealed five phosphoproteins that have molecular masses between 40 and 45 kilodaltons and are rapidly phosphorylated in response to epidermal growth factor of PDGF (4, 21). Each contains significant amounts of phosphotyrosine and phosphoserine, and in four of the five phosphoproteins lesser amounts of phosphothreonine are detectable (4). It seems that these five phosphoproteins collectively contain most of the phosphotyrosine in proteins of ca. 42 kilodaltons and thus account for a significant fraction of the rapidly phosphorylated tyrosine in epidermal growth factor-or PDGF-treated cells. Two of the five phosphoproteins (pp42A and pp42B) have identical SDS gel mobilities and are closely related to each other, as determined by one-dimensional peptide mapping (11). pp42B and a variable number of the other four phosphoproteins are phosphorylated at tyrosine under a variety of other conditions that lead to cell growth, including exposure to other growth factors (such as fibroblast growth factor) and nonpeptide mitogens (such as some tumor promoters) and transformation with certain retroviruses (1, 5-7, 11, 17, 18, 21; unpublished results). The correlation between mitogen addition and the appearance of pp42B is consistent with the notion that this phosphoprotein might be important for some event in the mitogenic response.We suspected that pp42B arises from a precursor protein through the actions of growth factor-activated and transformation-activated protein-tyrosine kinases and protein-serine or protein-threonine kinases. (24) was added to the labeling medium to a final concentration of 0.83 to 1.0 nM for 5 to 15 min. Cell fractionation procedures have been described before (8). Labeled cell cultures were prepared for twodimensional gel electrophoresis as described before (6,15). Samples of 0.01 ml were analyzed on isoelectric focusing gels as described before (15), except that the urea concentration was 9.15 M and the am...

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

“…One of the first phosphotyrosyl proteins to be identified in nontransformed cells was a 42-kDa protein, pp42 (6,9,19,35). pp42 is scarce (7) and evolutionarily conserved (5) and becomes tyrosine phosphorylated to high stoichiometry in response to diverse mitogens (2, 5-7, 9, 19, 24, 25, 35, 46 California, Berkeley, 1984). It has been previously hypothesized that pp42 is MAP-2 kinase, because MAP-2 kinase activity and pp42 copurify over several column steps (43).…”

Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.

“…Consistent with this suggestion, we found that tryptic phosphopeptide maps of MAP kinase yielded a single peptide, which corresponded to one ofthree major peptides generated by trypsin treatment of pp42 (ref. 2; data not shown).…”

“…Although numerous tyrosine-phosphorylated proteins have been identified by gel electrophoresis in oncogenically transformed or growth factor-stimulated cells, in most cases the biological significance of these phosphorylations has not been determined. pp42 is among the most widely studied of these tyrosine-phosphorylated proteins; it was identified by gel electrophoresis in cells stimulated by any of a number of diverse mitogens [epidermal growth factor (EGF), plateletderived growth factor, insulin-like growth factor II, thrombin, or phorbol 12-myristate 13-acetate (PMA; also called TPA)] (1)(2)(3)(4)(5)(6)(7) and also has been found in at least some oncogenically transformed cells (8)(9)(10). Because ofthe wide variety ofagents that stimulate this phosphorylation, pp42 is believed to be involved in some unknown step in intracellular signaling that is shared by all of these mitogens.…”

Kinases: positive and negative regulators of apoptosis

“…42,43 One substrate tyrosine phosphorylated in a number of different cell types, including hematopoietic cells and both T-and B-lymphocytes, [44][45][46] is a 42-kDa protein. [47][48][49] This protein was initially called MAP kinase. This name was based on the ability of this protein to phosphorylate microtubule associated protein.…”