The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences.

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180gag-fms encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180gag-fms) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120v-fms, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. Both constructs were biologically active when transfected into NIH 3T3 cells and produced morphologically transformed foci at equivalent efficiencies. When transfected into a cell line (psi 2) expressing complementary viral gene functions, G418-resistant (Neor) cells containing either of these vector DNAs produced high titers of transforming viruses. Analysis of proteins produced in cells containing the vector lacking gag gene sequences showed that gP180gag-fms was not synthesized, whereas normal levels of both immature gp120v-fms and mature gp140v-fms were detected. The glycoprotein was efficiently transported to the cell surface, and it retained wild-type tyrosine kinase activity. We conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180gag-fms is mediated by signal peptidase and that the amino termini of gp140v-fms and the c-fms gene product are identical.     

gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

Cells transformed by the McDonough strain of feline sarcoma virus express at their surface a v-fms-specific transmembrane glycoprotein designated gp140v-fms. By labeling with 32Pi, gp140v-fms was shown to be phosphorylated 30-fold more in serine residues than were the cytosolic v-fms polypeptides gp180gag-fms and gp120v-fms. By using the phosphotyrosine phosphatase-specific inhibitor sodium orthovanadate, an additional tyrosine phosphorylation was observed in vivo, again involving predominantly gp140v-fms. In vitro studies showed that the v-fms proteins were phosphorylated by protein kinase C in a calcium- and phosphatidylserine-dependent manner.     

Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage.

The McDonough strain of feline sarcoma virus contains an oncogene called v-fms whose ultimate protein product (gp140v-fms) resembles a cell surface growth factor receptor. To identify and characterize the protein product of the proto-oncogene c-fms, antisera were prepared to the viral fms sequences and used to detect specific cross-reacting sequences in human choriocarcinoma cells (BeWo) known to express c-fms mRNA. Both tumor-bearing rat sera and a rabbit antiserum prepared to a segment of v-fms expressed in Escherichia coli detected a 140-kilodalton (kDa) glycoprotein in the BeWo cells. Tryptic fingerprint analysis of [35S]methionine-labeled proteins indicated that the viral fms proteins and the 140-kDa BeWo cell protein were highly related. This 140-kDa glycoprotein contained an associated tyrosine kinase activity in vitro and was labeled principally on serine after 32Pi metabolic labeling. These results suggest that the 140-kDa protein in BeWo cells is the protein product of the human c-fms proto-oncogene. This conclusion is supported by the finding that a similar protein is detectable only in other human cells that express c-fms mRNA. These other human cells include adherent monocytes and the cell line ML-1, which can be induced to differentiate along the monocyte-macrophage pathway. This is in agreement with current thought that the c-fms proto-oncogene product functions as the CSF-1 receptor specific to this pathway.     

Colony-stimulating factor-1 receptor (c-fms)

The macrophage colony-stimulating factor, CSF-1 (M-CSF), is a homodimeric glycoprotein required for the lineage-specific growth of cells of the mononuclear phagocyte series. Apart from its role in stimulating the proliferation of bone marrow-derived precursors of monocytes and macrophages, CSF-1 acts as a survival factor and primes mature macrophages to carry out differentiated functions. Each of the actions of CSF-1 are mediated through its binding to a single class of high-affinity receptors expressed on monocytes, macrophages, and their committed progenitors. The CSF-1 receptor (CSF-1R) is encoded by the c-fms proto-oncogene, and is one of a family of growth factor receptors that exhibits an intrinsic tyrosine-specific protein kinase activity. Transduction of c-fms sequences as a viral oncogene (v-fms) in the McDonough (SM) and HZ-5 strains of feline sarcoma virus has resulted in alterations in receptor coding sequences that affect its activity as a tyrosine kinase and provide persistent signals for cell growth in the absence of its ligand. The genetic alterations in the c-fms gene that unmask its latent transforming potential abrogate its lineage-specific activity and enable v-fms to transform a variety of cells that do not normally express CSF-1 receptors.     

Transformation by the v-fms oncogene product: an analog of the CSF-1 receptor

The product of the c-fms proto-oncogene is related to, and possibly identical with, the receptor for the macrophage colony-stimulating factor, M-CSF (CSF-1). Unlike the product of the v-erbB oncogene, which is a truncated version of the EGF receptor, the glycoprotein encoded by the v-fms oncogene retains an intact extracellular ligand-binding domain so that cells transformed by v-fms express CSF-1 receptors at their surface. Although fibroblasts susceptible to transformation by v-fms generally produce CSF-1, v-fms-mediated transformation does not depend on an exogenous source of the growth factor, and neutralizing antibodies to CSF-1 do not affect the transformed phenotype. An alteration of the v-fms gene product at its extreme carboxyl-terminus represents the major structural difference between it and the c-fms-coded glycoprotein and may affect the tyrosine kinase activity of the v-fms-coded receptor. Consistent with this interpretation, tyrosine phosphorylation of the v-fms products in membranes was observed in the absence of CSF-1 and was not enhanced by addition of the murine growth factor. Cells transformed by v-fms have a constitutively elevated specific activity of a guanine nucleotide-dependent, phosphatidylinositol-4,5-diphosphate-specific phospholipase C. We speculate that the tyrosine kinase activity of the v-fms/c-fms gene products may be coupled to this phospholipase C, possibly through a G regulatory protein, thereby increasing phosphatidylinositol turnover and generating the intracellular second messengers diacylglycerol and inositol triphosphate.     

Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.     

Analysis of functional domains of the v-fms-encoded protein of Susan McDonough strain feline sarcoma virus by linker insertion mutagenesis.

The Susan McDonough strain of feline sarcoma virus contains an oncogene, v-fms, which is capable of transforming fibroblasts in vitro. The mature protein product of the v-fms gene (gp140fms) is found on the surface of transformed cells; this glycoprotein has external, transmembrane, and cytoplasmic domains. To assess the functional role of these domains in transformation, we constructed a series of nine linker insertion mutations throughout the v-fms gene by using a dodecameric BamHI linker. The biological effects of these mutations on the function and intracellular localization of v-fms-encoded proteins were determined by transfecting the mutated DNA into Rat-2 cells. Most of the mutations within the external domain of the v-fms-encoded protein eliminated focus formation on Rat-2 cells; three of these mutations interfered with the glycosylation of the v-fms protein and interfered with formation of the mature gp140fms. One mutation in the external domain led to cell surface expression of v-fms protein even in the absence of complete glycosylational processing. Cell surface expression of mutated v-fms protein is probably necessary, but is not sufficient, for cell transformation since mutant v-fms protein was found on the surface of several nontransformed cell lines. Mutations that were introduced within the external domain had little effect on in vitro kinase activity, whereas mutations within the cytoplasmic domain all had strong inhibitory effects on this activity.     

Appropriate glycosylation of the fms gene product is a prerequisite for its transforming potency.

Processing inhibitors of N-linked glycans were used to determine whether correct glycosylation of the oncogene product gp140v-fms, encoded by the McDonough strain of feline sarcoma virus (SM-FeSV), is required to maintain the oncogenic properties of v-fms. SM-FeSV-transformed cells treated with the glucosidase-I inhibitors N-methyldeoxynojirimycin (MdN) or castanospermine synthesized predominantly a gp125v-fms species which had a normal half life. The molecule was transported to the plasma membrane and exhibited normal kinase activity as determined by autophosphorylation. However, although no significant change in cell morphology of the SM-FeSV-transformed cells was observed in the presence of castanospermine, growth of these cells became strictly serum-dependent. In addition, growth in soft agar was drastically retarded despite the presence of 10% calf serum, indicating that the transformed properties of the cells were altered. In contrast, swainsonine, an inhibitor of the processing alpha-mannosidase-II, had no effect. Cells transformed by the Snyder Theilen strain of FeSV were used to demonstrate that the altered proliferative properties were directly linked to the modified structure of the fms gene product. Our data suggest that the extracellular domain of gp140v-fms plays a role in regulating cell proliferation.     

Transformation by the v-fms oncogene product: role of glycosylational processing and cell surface expression.

The effect of glycosylational-processing inhibitors on the synthesis, cell surface expression, endocytosis, and transforming function of the v-fms oncogene protein (gp140fms) was examined in McDonough feline sarcoma virus-transformed Fischer rat embryo (SM-FRE) cells. Swainsonine (SW), a mannosidase II inhibitor, blocked complete processing, but an abnormal v-fms protein containing hybrid carbohydrate structures was expressed on the cell surface. SW-treated SM-FRE cells retained the transformed phenotype. In contrast, two glucosidase I inhibitors (castanospermine [CA] and N-methyl-1-deoxynojirimycin [MdN]) blocked carbohydrate remodeling at an early stage within the endoplasmic reticulum and prevented cell surface expression of v-fms proteins. CA-treated SM-FRE cells reverted to the normal phenotype. Neither SW, CA, nor MdN affected either endocytosis or the tyrosine kinase activity associated with the v-fms gene product in vitro. These results demonstrate the necessity of carbohydrate processing for cell surface expression of the v-fms gene product and illustrate the unique ability to modulate the transformed state of SM-FRE cells with the glycosylational-processing inhibitors CA and MdN.     

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.     

Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.     

McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms).

The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P170gag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (P120fms and pp55gag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 +/- 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRI and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Synder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline-leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes.     

The unique insert of cellular and viral fms protein tyrosine kinase domains is dispensable for enzymatic and transforming activities.

The receptors for colony stimulating factor-1 (CSF-1), platelet derived growth factor and the c-kit protein tyrosine kinase (PTK) contain within their catalytic domains a stretch of 60-100 residues, largely unrelated in sequence, with no counterpart in other PTKs. Of the 64 amino acids within this kinase insert, 58 were deleted from the mouse CSF-1 receptor by oligonucleotide-directed mutagenesis. The mutant CSF-1 receptor was not markedly affected in its kinase activity, post-translational processing or its ability to induce autocrine transformation of NIH 3T3 mouse fibroblasts. Similarly, retention of kinase and transforming activities were observed following deletion of part or all of the kinase insert from the v-fms oncoprotein. The c- and v-fms kinase inserts were probed using monoclonal and polyclonal antibodies and were found to be highly antigenic. Two monoclonal antibodies raised to the v-fms cytoplasmic domain both recognized epitopes within the insert, and bound enzymatically active v-fms glycoproteins. These results indicate that the fms kinase insert is located on the surface of the protein and folds separately from the rest of the catalytic domain, but is not required for the biological activity of fms PTKs ectopically expressed in mouse fibroblasts. The insert may therefore play a specific function in cells such as monocytes and trophoblasts that normally express the CSF-1 receptor.     

Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.     

Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of the RAF-1 proto-oncogene product.

The serine/threonine kinase RAF-1 is phosphorylated in intact macrophages in response to CSF-1 at 37 degrees C The augmented phosphorylation of RAF-1 and a concomitant increase in RAF-1 associated serine/threonine kinase activity are kinetically later events than CSF-1 induced protein tyrosine phosphorylation. Furthermore, phosphoamino acid analysis of RAF-1 reveals the presence of phosphoserine, trace amounts of phosphothreonine but no phosphotyrosine and the phosphorylated RAF-1 does not react with anti-phosphotyrosine antibodies. In contrast to CSF-1 induced protein tyrosine phosphorylation, RAF-1 phosphorylation and activation are temperature dependent and do not occur at 4 degrees C. Furthermore, coprecipitation experiments failed to reveal any noncovalent association of RAF-1 with the CSF-1 receptor. Thus, while RAF-1 is not a direct substrate for the CSF-1 receptor tyrosine kinase in vivo, its temperature dependent phosphorylation and activation represent an intriguing aspect of the CSF-1 response.     

c-kit原癌基因两个相邻EcoRI片段的核苷酸序列和结构特点

 c-kit原癌基因(Proto-oncogene)是猫逆转录病毒HZ4 FeSV v-kit癌基因的类似物,其结构与CSF-1R和PDGFR十分相似,基因产物是一种跨膜糖蛋白,具有酪氨酸蛋白激酶活性,细胞外部分则可能是一种受体,可接受细胞外信号的调节。 本文报道人c-kit原癌基因两个相邻片段的核苷酸序列及其结构特点。     

Characterization of the insulin receptor kinase purified from human placental membranes

The insulin receptor purified from human placenta by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose to near homogeneity retained tyrosine-specific protein kinase activity. This purified insulin receptor kinase specifically catalyzed the incorporation of 32P from [gamma-32P]ATP into not only the beta-subunit of the insulin receptor but also histone H2B, a synthetic peptide which is sequentially similar to the site of tyrosine phosphorylation in pp60src (a gene product of the Rous sarcoma virus) and antibodies to pp60src present in the sera obtained from three rabbits bearing tumors induced by the Rous sarcoma virus. In each case, phosphorylation occurred exclusively on tyrosine residues. Insulin stimulated phosphorylation of these substrates 3- to 5-fold. Kinetic analysis using the synthetic peptide indicated that insulin acted by increasing the Vmax of peptide phosphorylation from about 3.1 to 9.5 nmol X mg-1 of protein X min-1, whereas the value of the Km for the peptide, about 1.5 mM, was not significantly changed. This kinase acted weakly on casein, alpha-S-casein, actin, and a tyrosine-containing peptide analogue of a serine-containing peptide used commonly as a substrate for the cyclic AMP-dependent protein kinases. These data show that the insulin receptor kinase displays specificity toward exogenous substrates similar to the substrate specificity observed for pp60src and the protein kinase activity associated with the receptor for epidermal growth factor. The data suggest that the catalytic sites of these three tyrosine kinases are similar and that insulin activates its receptor kinase by increasing the Vmax.     

Monoclonal antibodies to the transformation-specific glycoprotein encoded by the feline retroviral oncogene v-fms.

Monoclonal antibodies prepared to epitopes encoded by the transforming gene (v-fms) of the McDonough strain of feline sarcoma virus were used to study v-fms-coded antigens in feline sarcoma virus-transformed rat and mink cells. These antibodies reacted with three different polypeptides (gP180gag-fms, gp140fms, and gp120fms), all of which were shown to be glycosylated. Protein blotting with [125I]-labeled monoclonal immunoglobulin G's was used to determine the relative steady-state levels of these glycoproteins in transformed cells and showed that gp120 and gp140 were the predominant products. Immunofluorescence assays and subcellular fractionation experiments localized these molecules to the cytoplasm of transformed cells in quantitative association with sedimentable organelles. Thus, v-fms-coded glycoproteins differ both chemically and topologically from the partially characterized products of other known oncogenes and presumably transform cells by a different mechanism.     

Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C

The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor beta subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled beta subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)