Mitogen‐activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P‐labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two‐dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision‐activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1‐ and KSS1‐encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.

ABSTRACTpp42, a low-abundance 42-kDa protein, becomes transiently phosphorylated on tyrosine after stimulation of fibroblasts by a variety of mitogens, including epidermal growth factor, platelet-derived growth factor, phorbol 12-myristate 13-acetate, thrombin, and insulin-like growth factor II. The induction of pp42 phosphorylation on tyrosine by such diverse mitogenic agents suggests an important role for pp42 in the cascade of events necessary for cell transition from Go into the cell cycle. However, as with most proteins identified on the basis of their tyrosine phosphorylation, the function of pp42 in cellular regulation is unknown. In this manuscript we report evidence that suggests that pp42 is a serine/threonine-specific protein kinase. Stimulation of 3T3-L1 cells with insulin has been shown to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (MAP-2) and ribosomal protein S6 kinase H. This cytosolic serine/threonine protein kinase, which itself is phosphorylated on tyrosine, has been termed "MAP kinase." We now report that pp42 phosphorylation and MAP kinase activation occur in fibroblasts in response to similar mitogens, that the two proteins comigrate on one-and two-dimensional polyacrylamide gels, and that the two proteins copurify chromatographically. The major peptides generated from purified MAP kinase by V8 protease digestion are present as a subset of the peptides in digests of pp42 excised from two-dimensional gels. Thus, the results suggest that MAP kinase is tyrosine-phosphorylated pp42.Many growth-factor receptors and oncogene products are tyrosine protein kinases. To understand the mechanisms of intracellular signaling used by these kinases, it will be important to identify and characterize their cellular substrate proteins. 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)](1-7) and also has been found in at least some oncogenically transformed cells (8-10). 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.Recently a novel serine/threonine protein kinase was identified in insulin-stimulated 3T3-L1 cells (11). This protein kinase has been shown to phosphorylate microtubuleassociated protein 2 (MAP-2) as well as ribosomal protein S6 kinase II in vitro (12). The kinase was also found to migrate on sodium dodecyl sulfate (SDS)/polyacrylamide gels with a molecular ...

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp6)v-slC are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp6OV-slc altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-y, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp6OV-sC and the EGF receptor, phospholipase C-,y was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60's'-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.The v-src oncogene product, pp60v-sr, is a 60-kDa tyrosyl protein kinase, and this enzymatic activity is essential to its ability to cause malignant transformation (52). Numerous cellular proteins become phosphorylated on tyrosine in src-transformed cells (9,34,35,45), but the mechanism(s) by which these src-induced phosphorylations alter cellular behavior is unknown. Studies utilizing mutant forms of pp60v-src which retain kinase activity but are partially defective in their ability to cause phenotypic transformation suggest that only a limited subset of these tyrosine-phosphorylated proteins play an essential role in cellular transformation by src (10,33,35,39,57). It is widely suspected that the physiologically important substrates for pp60v-src will prove to be proteins which play a role in normal growth regulation, although it has been difficult to identify pp60v-src_ induced changes in phosphorylation and function of such regulatory proteins. It also is generally believed that at least some critical substrates for pp60v-sr" are associated with cell membranes, since cytosolic variants of pp60v-src are unable to transform cells (33,39,52,57).The best-characterized membrane-associated growth regulatory proteins are the transmembrane receptors for growth factors, and we have hypothesized (49) that pp60v-src might alter cellular regulation by phosphorylating and activating growth factor receptors. Indeed, we recently found that a 95-kDa cellular glyco...