The molybdate-stabilized glucocorticoid binding complex of L-cells contains a 98-100 kdalton steroid binding phosphoprotein and a 90 kdalton nonsteroid-binding phosphoprotein that is part of the murine heat-shock complex

This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabilized cytosol prepared from 32P-labeled L-cells. In our early work, we showed that cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90 and a 98-100 kdalton protein) that elute from an affinity resin of deoxycorticosterone agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor. Both phosphoproteins are immunoadsorbed onto protein-A-Sepharose from molybdate-stabilized cytosol incubated with a monoclonal antibody against the receptor. The 98-100 kdalton phosphoprotein binds steroid and the 90 kdalton phosphoprotein is a structurally different, nonsteroid-binding protein that is bound to the untransformed, molybdate-stabilized glucocorticoid receptor. The 90 kdalton protein reacts on Western blots with a monoclonal antibody raised against a 90 kdalton protein from the water mold Achlya ambisexualis. This antibody recognizes an epitope that is conserved in 90 kdalton phosphoproteins from rodent and human cells, and it reacts with the 90 kdalton phosphoprotein that copurifies with the molybdate-stabilized, untransformed chick oviduct progesterone receptor. The 90 kdalton nonsteroid-binding phosphoprotein is an abundant cytosolic protein that dissociates from the glucocorticoid receptor when it is transformed, and unlike the steroid-binding protein, it does not bind to DNA. The 90 kdalton phosphoprotein determines the acidic behavior of the untransformed glucocorticoid receptor on DEAE-cellulose. This abundant cytosolic 90 kdalton phosphoprotein reacts with rabbit antiserum raised against the gel purified 89 kdalton chicken heat-shock protein (hsp89). This antiserum recognizes 90 kdalton heat-shock proteins in human, rodent, frog and Drosophila cells. Immunoadsorption of molybdate-stabilized cytosol with antibody directed against the 98-100 kdalton steroid receptor results in the immune-specific adsorption of a 90 kdalton phosphoprotein that reacts with anti-hsp89 antibody on Western blots. These observations suggest that, like the transforming proteins from several avian sarcoma viruses, the untransformed glucocorticoid receptor exists in a complex with the 90 kdalton heat-shock protein.     

Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.

The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.     

Identification and characterization of cellular targets for tyrosine protein kinases

Protein kinases associated with the transforming proteins of a number of retroviruses are specific for tyrosine. Several proteins in cells transformed by these viruses are phosphorylated at tyrosine. We have now identified three unrelated abundant nonphosphorylated cellular proteins of 46,000, 39,000 and 28,000 daltons in chick embryo cells, which are the unphosphorylated forms of phosphotyrosine-containing proteins and thus are substrates for tyrosine protein kinases. By two-dimensional gel analysis, we have found that the 46,000-dalton protein exists in two unphosphorylated forms of which the more acidic is a minor species. This latter form is phosphorylated, chiefly at serine, in both normal and transformed cells, generating a yet more acidic species. In transformed but not normal cells, the major form is phosphorylated at tyrosine and serine, yielding a fourth isoelectric variant. The 46,000-dalton unphosphorylated protein has been partially purified, and antiserum to it recognizes all four isoelectric variants of the protein. The 39,000-dalton protein has two unphosphorylated forms of which the more acidic is a minor species. The major form is phosphorylated at tyrosine and serine in transformed cells only. The 39,000-dalton unphosphorylated protein has been partially purified, and antiserum raised to it recognizes all three isoelectric variants. The 28,000-dalton protein has a single phosphorylated form which contains serine in normal cells, but both serine and tyrosine in transformed cells.     

Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.     

Identification and characterization of the major chicken bone phosphoprotein. Analysis of its synthesis by cultured embryonic chick osteoblasts

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O-[32P]phosphoserine and O-[32P]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major approximately 66-kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7 - 3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical approximately 66-kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE-Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino-terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine-residue sequence spanning the Arg-Gly-Asp cell-binding site of the rat protein osteopontin, was identified in the approximately 66-kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compositions. Several smaller-molecular-mass phosphoproteins ranging in size over about 55 - 29 kDa were also observed in the HCl extracts of bone. Peptide mapping of these species demonstrated that the approximately 66-kDa, approximately 55-kDa, and approximately 45-kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the approximately 66-kDa species since this phosphoprotein remained the prevalent species after a 24-h chase. Because of the predominant association of all the smaller-molecular-mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24-h chase, these results suggested that the lower-molecular-mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the approximately 66-kDa component in the extracellular matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that the 90-kDa phosphoprotein associated with the untransformed L-cell glucocorticoid receptor is a murine heat shock protein

Two phosphoproteins are adsorbed to protein-A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98-100-kDa phosphoprotein that contains the steroid-binding site and the other is a 90-kDa nonsteroid-binding phosphoprotein that is associated with the untransformed, molybdate-stabilized receptor (Housley, P. R., Sanchez, E. R., Westphal, H.M., Beato, M., and Pratt, W.B. (1985) J. Biol. Chem. 260, in press). In this paper we show that the 90-kDa receptor-associated phosphoprotein is an abundant cytosolic protein that reacts with a monoclonal antibody that recognizes the 90-kDa phosphoprotein that binds steroid receptors in the chicken oviduct. The 90-kDa protein immunoadsorbed from L-cell cytosol with this antibody reacts on Western blots with rabbit antiserum prepared against the 89-kDa chicken heat shock protein. Immunoadsorption of molybdate-stabilized cytosol by antibodies against the glucocorticoid receptor results in the presence of a 90-kDa protein that interacts on Western blots with the antiserum against the chicken heat shock protein. The association between the 90-kDa protein and the receptor is only seen by this technique when molybdate is present to stabilize the complex; and when steroid-bound receptors are incubated at 25 degrees C to transform them to the DNA-binding state, the 90-kDa protein dissociates. These observations are consistent with the proposal that the untransformed glucocorticoid receptor in L-cells exists in a complex with the murine 90-kDa heat shock protein.     

Purification and biochemical characterization of statin, a nonproliferation specific protein from rat liver

The nuclear protein statin, detectable with specific monoclonal antibodies, is found mostly in nonproliferating cells (Wang, E. (1985) J. Cell Biol. 100, 545-551). In the rat liver a 57-kDa protein designated as rat liver protein 57 (RLp57) was recently identified to carry the epitope for the anti-statin-specific monoclonal antibody, S-44 (Sester, U., Moutsatsos, I. K., and Wang, E. (1989) Exp. Cell Res. 182, 550-558). To characterize further the RLp57 protein, in the present study a polyclonal antibody was raised to the RLp57 protein eluted from polyacrylamide gel. Similar to the anti-statin monoclonal antibody, this polyclonal antibody recognizes a nuclear antigen in nonproliferating fibroblasts and reacts with a 57-kDa protein in rat liver and nonproliferating cells strongly suggesting that RLp57 is a statin protein from rat liver. Two isoforms of RLp57 (isoelectric points between 6.5 and 7.0) were detected after two-dimensional gel electrophoresis and immunoblotting. RLp57 was purified using multiple chromatographic steps, including ion-exchange and affinity chromatography followed by chromatofocusing. These results show that RLp57, a statin protein found in liver, has two isoelectric variants and can be purified to apparent homogeneity by sequential steps of chromatographic procedures.     

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

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.     

Alterations in the transferrin receptor of human erythroleukemic cells after induction of hemoglobin synthesis

When K562 human erythroleukemic cells are induced to differentiate by addition of hemin to their medium, the number of binding sites for transferrin on the cell surface is substantially reduced. This reflects an internalization of receptors since no such reduction is observed when the total binding sites in soluble extracts of uninduced and differentiating cells are compared. The internalization of transferrin receptors has also been shown using lactoperoxidase-mediated radioiodination of cell surfaces and by immune precipitation of total and surface labeled receptors using an anti-receptor monoclonal antibody. Transferrin receptors from uninduced and differentiating cells were partially purified by affinity chromatography on transferrin-Sepharose and shown to be disulfide-bridged homodimers of a polypeptide with an apparent molecular weight of approximately 90,000. This protein is a phosphoprotein that can be resolved by isoelectric focusing into three major and two minor forms. By digestion with bacterial alkaline phosphatase, it was shown that at least four of these forms are probably phosphorylation variants of a single polypeptide. As differentiation proceeds, the proportions of the individual forms of the receptor change with a shift to the more phosphorylated polypeptides.     

Calmodulin and Ca2+-dependent phosphorylation and dephosphorylation of 63-kDa subunit-containing bovine brain calmodulin-stimulated cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes which are composed of two distinct subunits: Mr 60,000 and 63,000. The 60-kDa but not the 63-kDa subunit-containing isozyme can be phosphorylated by cAMP-dependent protein kinase resulting in decreased affinity of this subunit toward calmodulin (Sharma, R. K., and Wang, J. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2603-2607). In contrast, purified 63-kDa subunit-containing isozyme has been found to be phosphorylated by a preparation of bovine brain calmodulin-binding proteins in the presence of Ca2+ and calmodulin. The phosphorylation resulted in the maximal incorporation of 2 mol of phosphate/mol of the phosphodiesterase subunit with a 50% decrease in the enzyme affinity toward calmodulin. At a constant calmodulin concentration of 6 nM, the phosphorylated isozyme required a higher concentration of Ca2+ for activation than the nonphosphorylated phosphodiesterase. The Ca2+ concentrations at 50% activation by calmodulin of the nonphosphorylated and phosphorylated isozymes were 1.1 and 1.9 microM, respectively. Phosphorylation can be reversed by the calmodulin-dependent phosphatase, calcineurin, but not by phosphoprotein phosphatase 1. The results suggest that the Ca2+ sensitivities of brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes can be modulated by protein phosphorylation and dephosphorylation mechanisms in response to different second messengers.     

Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells

Glucocorticoid-sensitive L-cells were cultured for 18 h in the presence of [32P]orthophosphate and steroid-binding proteins of cytosol were separated by affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cytosol contains a major phosphoprotein of Mr = 92,000 and a minor phosphoprotein of Mr = 100,000, both of which bind glucocorticoids in a stereospecific, high affinity manner and have the same Mr as glucocorticoid receptor species that have been covalently labeled with the site-specific affinity ligand [3H] 9 alpha-fluoro-16-methyl-11 beta,17 alpha,21-trihydroxypregna-1, 4-diene-3,20-dione 21-mesylate. Cytosol from 32P-labeled, glucocorticoid-resistant L-cells possessing 5% of the steroid-binding capacity of sensitive cells contains very little of the Mr = 92,000 phosphoprotein and none of the Mr = 100,000 phosphoprotein. These observations provide strong evidence that the glucocorticoid receptor is phosphorylated by intact L-cells. The Mr = 92,000 protein is phosphorylated on serine and it can be resolved into two species using isoelectric focusing, consistent with the proposal that there is more than 1 phosphorylated serine/steroid-binding unit. The glucocorticoid-resistant L-cell line produces a unique phosphoprotein of Mr = 104,000 that is recovered in variable amounts after affinity chromatography. It is not known whether this phosphoprotein is a separate gene product or whether it represents a precursor with weak steroid-binding activity that is not cleaved in the resistant cell to the high affinity, Mr = 92,000 mature receptor form.     

Normal rat kidney cells secrete both phosphorylated and nonphosphorylated forms of osteopontin showing different physiological properties

We have reported previously that the 69-kDa major phosphoprotein, secreted by normal rat kidney (NRK) cells, is osteopontin, a glycosylated bone matrix protein. Here we show that this 69-kDa osteopontin is secreted by NRK cells in both phosphorylated (pp69) and nonphosphorylated (np69) forms, with estimated isoelectric points of 3.8 and 4.5, respectively. Electrophoretic analysis of radioiodinated cell surface proteins immunoprecipitated with an anti-69-kDa osteopontin serum, demonstrates that the 69-kDa osteopontin is also present on the cell surface, but only its phosphorylated form (pp69) shows such cell surface association. Because osteopontin mediates cell adhesion and spreading, and contains an Arg-Gly-Asp-Ser cell-binding sequence, our observations strongly suggest that the cell surface localization of pp69 osteopontin is receptor-mediated, and the modification by phosphorylation may be crucial for its receptor binding activity. We also report that antisera directed against either fibronectin or 69-kDa osteopontin co-immunoprecipitate both np69 osteopontin and fibronectin as a heat-dissociable complex. In contrast, pp69 osteopontin does not co-precipitate with fibronectin. These observations demonstrate an interactive relationship between np69 and soluble fibronectin. Furthermore, compared to NRK cells, vanadyl sulfate-treated NRK cells which acquire a reversible transformed phenotype, including anchorage-independent growth, show increased levels of pp69 on the cell surface, concomitant with significantly decreased levels of pp69 and elevated levels of np69 in the conditioned media. The data presented here establish transformation sensitivity of NRK cell-secreted osteopontin with respect to its secretion and cell surface localization, and demonstrate that phosphorylated and nonphosphorylated forms of osteopontin have different physiological properties, which may regulate the functional roles of this extracellular matrix protein.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle alpha subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that alpha211, the entire amino-terminal extracellular domain of AChR alpha subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The alpha211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mm ( approximately 30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with alpha-bungarotoxin (k(D) 0.2 nm) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of alpha211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently (15)N-labeled in P. pastoris, and proton cross-peaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.     

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.     

Purification of a heterodimeric betaine aldehyde dehydrogenase from wild amaranth plants subjected to water deficit

Betaine aldehyde dehydrogenase was purified to homogeneity from wild-type amaranth plants subjected to water deficit. The enzyme has a native molecular mass of 125 kDa; it is formed by two subunits, one of the subunits with a molecular mass of 63 kDa and the second one of 70 kDa as determined by SDS-PAGE and double dimension electrophoresis. IEF studies showed two bands with pI values of 4.93 and 4.85, respectively. Possible glycosilation of the 63- and 70-kDa subunits were tested with negative results. Both subunits cross-reacted strongly with polyclonal antibody raised against porcine kidney BADH. Also antiserum rose against HSP70 cross-reacted strongly with the wild amaranth BADH 70-kDa subunit. The enzyme was stable to extreme pH's and temperatures, and high KCl concentrations. Product inhibition of BADH was not observed.     

Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils

The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.