Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C.

The postsynaptic glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C. The target for phosphorylation is the strychnine-binding subunit of the receptor (molecular mass of approximately 48 kDa), which is phosphorylated on serine residues to a final stoichiometry of approximately 0.8 mol of phosphate/mol of subunit. The 48-kDa phosphoprotein was analyzed by proteolytic cleavage and peptide mapping in order to localize the site of phosphorylation within the receptor molecule. Examination of the 32P-labeled receptor fragments generated by digestion with N-chlorosuccinimide, cyanogen bromide, and endoproteinase lysine C and of the deduced amino acid sequence of the 48-kDa protein (Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215-220) indicates that the phosphorylation site is located in a region corresponding to the major intracellular loop of the predicted structure of the glycine receptor subunit and suggests serine 391 as the phosphorylated residue. In fact, a synthetic peptide corresponding to residues 384-392 of the 48-kDa subunit was specifically phosphorylated by protein kinase C. Moreover, tryptic digests of this phosphopeptide and of the phosphorylated 48-kDa subunit of the glycine receptor migrated to the same position in two-dimensional peptide mapping. Furthermore, antibodies elicited against peptide 384-392 were shown to inhibit the protein kinase C-dependent phosphorylation of the 48-kDa polypeptide. Interestingly, the relative position of the phosphorylated domain is similar to those known or proposed to be phosphorylated in other ligand-gated ion channel receptor subunits, thus suggesting further the existence of a homologous regulatory region in these receptor proteins.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

Type I and Type II γ-Aminobutyric Acid/Benzodiazepine Receptors: Purification and Analysis of Novel Receptor Complex from Neonatal Cortex

The gamma-aminobutyric acid (GABA) type A receptor was purified several thousandfold by affinity chromatography from rat cerebellum, adult cortex, and neonatal cortex. Competition for the benzodiazepine binding site by CL 218872 indicated that cerebellar receptors were predominantly type I, adult cortical receptors were a mixture of subtypes, and neonatal cortex was enriched in type II receptor. The receptor purified from neonatal cortex contained predominantly a 54-kilodalton (kDa), beta-subunit-like protein, whereas receptors from cerebellum and adult cortex contained nearly equal amounts of a 50-kDa, alpha-subunit-like protein and a 54-kDa polypeptide. Peptide maps of trypsin-digested 54-kDa subunits from cerebellum, adult cortex, and neonatal cortex exhibited very similar profiles, a result indicating considerable homology between these proteins in the receptor subtypes. A 59-kDa subunit protein was detected in the receptor complex purified from neonatal cortex. Like the 50-kDa, alpha-subunit of the type I receptor, this protein was photolabeled with [3H]flunitrazepam. The photolabeled peptide fragments, produced by trypsin digestion of these alpha 50- and alpha 59-subunits, exhibited the same retention times on reverse-phase HPLC. A less highly purified GABAA receptor preparation from adult rat spinal cord possessed characteristics that were very similar to those of the receptors purified from neonatal cortex.     

Evidence for a structurally homologous Rh-like polypeptide in Rhnull erythrocytes.

Human Rhnull red blood cells fail to react with Rh antibodies, indicating that these cells are either devoid of Rh protein or, like other species, possess antigenically distinct variants. To determine whether Rhnull cells possess an Rh-like polypeptide, 32-kDa proteins from D--, rr, and Rhnull cells were labeled with the cysteine-specific probe, 125I-labeled pyridyldithioethylamine. Size comparisons of labeled proteins in Triton X-100-solubilized membranes from Rh-bearing and Rhnull cells showed similar sedimentation coefficients and Stoke's radii. Immunoprecipitated Rh(D) from D-- cells, Rh(c) from rr cells, and purified 32-kDa proteins from Rhnull cells were digested with alpha-chymotrypsin and examined by high-performance liquid chromatography and by two-dimensional iodopeptide mapping. Analysis of 125I-labeled chymotryptic fragments from immunoprecipitated Rh(D) and Rh(c) showed the labeled peptides from both phenotypes to be virtually identical. High-performance liquid chromatography profiles and iodopeptide maps of 32-kDa Rhnull proteins yielded patterns identical to 32-kDa proteins isolated from D-- cells and rr cells with the exception of one missing 125I-labeled peptide. Further analysis of the Rh-related fragments from Rhnull cells showed significant homology with immunoprecipitated Rh(D) and Rh(c). DNA sequence analysis of cysteine-encoding regions from Rh-bearing and Rhnull cells showed complete identity. These data suggest that Rhnull red blood cells, although serologically distinct, possess an Rh-like protein that is structurally very similar to Rh(D) and Rh(c).     

Amino acid sequence of protein B23 phosphorylation site

A major phosphopeptide labeled in vivo, was identified in nucleolar protein B23 (Mr/pI = 37,000/5.1) after tryptic digestion. This peptide was purified by high performance liquid chromatography using reverse-phase (C8 and C18) columns. The phosphopeptide contains 20 amino acids including 1 phosphoserine, 7 glutamic acids, and 4 aspartic acids. The amino acid sequence is: His-Leu-Val-Ala-Val-Glu-Glu-Asp-Ala-Glu-Ser(P)-Glu-Asp-Glu-Asp- Glu-Glu-Asp-Val-Lys. This amino acid sequence is similar to that of nucleolar phosphoprotein C23 (8 consecutive amino acids were identical), and to the regulatory subunit (RII) of cAMP-dependent protein kinase (7 consecutive amino acids were identical, which is phosphorylated by casein kinase II (Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982) J. Biol. Chem 257, 10440-10445). The regions near these phosphorylation sites are enriched with glutamic and aspartic acids, suggesting that this acidic amino acid cluster may be essential for kinase recognition.     

Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.     

A basic NH2-terminal extension of rat liver arginyl-tRNA synthetase required for its association with high molecular weight complexes

Rat liver arginyl-tRNA synthetase is found in extracts either as a component (Mr = 72,000) of a high molecular weight aminoacyl-tRNA synthetase complex or as a low molecular weight (Mr = 60,000) free form. Previous studies suggested that the free protein arises from the complex-derived form by a limited proteolysis that removes the portion of the protein required for its association with the complex. In order to determine the location in the protein and some structural properties of this extra 12-kDa portion, the complex-derived and free forms were each extensively purified and compared by peptide mapping using limited V-8 protease digestion. The two proteins showed 7-8 peptide bands in common, as well as 1-2 unique bands each. Treatment of each of the proteins with carboxypeptidase Y prior to digestion with V-8 protease indicated that the two proteins have a common COOH-terminal peptide. Amino acid analyses of the two arginyl-tRNA synthetases revealed a strong similarity; however, the complex-derived form contained a large excess of basic amino acids. These results demonstrate directly that the complex-derived and free forms of arginyl-tRNA synthetase are closely related proteins, but that the former includes a basic, NH2-terminal extension absent in the free form. The role of this extra segment in the polyanion-binding properties of eukaryotic synthetases and in their structural organization into high molecular weight complexes is discussed.     

A protein tyrosine kinase involved in regulation of pp60c-src function

We recently identified a novel protein tyrosine kinase that specifically phosphorylates truncated pp60c-src (Mr = 53,000) at a tyrosine residue(s) distinct from its autophosphorylation site. In this study, we examined whether this enzyme phosphorylates intact pp60c-src (Mr = 60,000) and determined its phosphorylation site. Non-neuronal and neuronal forms of intact pp60c-src were separately purified from the membrane fraction of neonatal rat brain by sequential column chromatographies. The novel kinase phosphorylated tyrosine residues of both forms of intact pp60c-src. The phosphorylation occurred in parallel with autophosphorylation of pp60c-src, and in both forms the final stoichiometry estimated was quite similar to that of autophosphorylation (about 5%). The enzyme also phosphorylated pp60c-src in which the kinase activity had been destroyed by an ATP analogue, p-fluorosulfonylbenzoyl 5'-adenosine. The phosphorylation site of the non-neuronal form was analyzed by sequential peptide mapping with tosylphenylalanyl chloromethyl ketone-treated trypsin and alpha-chymotrypsin. Tryptic digestion of the phosphorylated pp60c-src yielded a unique phosphopeptide that cross-reacted with an antibody specific for the carboxyl-terminal sequence of chicken pp60c-src. Digestion of the phosphopeptide with chymotrypsin yielded a product that comigrated with a synthetic phosphopeptide corresponding to the carboxyl-terminal 15 residues of chicken pp60c-src. These results clearly indicate that the carboxyl-terminal sequence of rat pp60c-src is identical to that of chicken pp60c-src, and a tyrosine residue corresponding to chicken Tyr527 is the phosphorylation site. This phosphorylation resulted in a decrease in the enolase phosphorylating activity of pp60c-src. Kinetic experiments indicated that this decrease in activity was due to a decrease in the Vmax value of pp60c-src. These findings support our previous proposal that the novel tyrosine kinase acts as a specific regulator of pp60c-src in cells.     

Evidence for a single steroid-binding protein in the rabbit progesterone receptor

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.     

Photoaffinity probes for the alpha 1-adrenergic receptor and the calcium channel bind to a common domain in P-glycoprotein

P-glycoprotein is a 130-180-kDa integral membrane protein that is overproduced in multidrug-resistant cells. The protein appears to act as an energy-dependent drug efflux pump that has broad specificity for structurally diverse hydrophobic antitumor drugs. Many agents, such as the calcium channel blocker verapamil, reverse multidrug resistance and also interact with P-glycoprotein. The goal of this work was to determine if a common binding site participates in the transport of antitumor drugs and/or the reversal of drug resistance. This was done by comparing the peptide maps of P-glycoprotein (encoded by mdr1b) after it was labeled with a photoactive calcium channel blocker, [3H]azidopine, and a newly identified photoaffinity analog for P-glycoprotein 2-[4-(4-azido-3-[125I]iodobenzoyl) piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline [( 125I]iodoaryl azidoprazosin). [125I] Iodoaryl azidoprazosin, which classically has been used to identify the alpha 1-adrenergic receptor, bound to P-glycoprotein and was preferentially competed by vinblastine greater than actinomycin D greater than doxorubicin greater than colchicine. Peptide maps derived from P-glycoprotein labeled with [3H]azidopine or [125I]iodoaryl azidoprazosin were identical. After maximal digestion under conditions for Cleveland mapping, a single major 6-kDa fragment was obtained after digestion with V8 protease, whereas two major fragments, 6.5 and 5.5 kDa, were detected after digestion with chymotrypsin. The 6.0-kDa V8 fragment and the 6.5-kDa chymotrypsin fragment were both found when P-glycoprotein encoded by mdr1a and mdr1b was compared. Despite its specific interaction with P-glycoprotein, neither iodoaryl azidoprazosin nor prazosin markedly reversed resistance compared with verapamil or azidopine. Further, multidrug-resistant cells were 900-fold resistant to vinblastine but only 5-fold resistant to prazosin. These data demonstrate that structurally diverse reversal and/or antitumor agents are likely to have differential affinity for a small common domain of P-glycoprotein.     

Identification of myosin II kinase from sea urchin eggs as protein kinase CK2

Here we purified and identified a myosin II kinase from sea urchin eggs. The activity of this myosin II kinase in the egg extract was not significantly affected by Ca(2+)/calmodulin (CaM). Using sequential column chromatographies, we purified the myosin II kinase from the egg extract as a complex composed of 36- (p36) and 28-kDa (p28) proteins. Partial amino acid sequences of these two components were highly coincident with those of the alpha and beta subunits of protein kinase CK2 (formerly casein kinase II) in sea urchin eggs, respectively. To confirm that the purified myosin II kinase was CK2, we obtained a cDNA which encodes p36 from a cDNA library of sea urchin eggs. The amino acid sequence derived from the obtained cDNA showed over 70% homology to CK2 from various eukaryotes. Furthermore, recombinant p36, as well as the purified myosin II kinase, phosphorylated MRLC. One dimensional phosphopeptide mapping revealed that the phosphorylation site(s) of MRLC by both recombinant p36 and the purified myosin II kinase was identical. These clearly showed that the Ca(2+)/CaM-independent myosin II kinase activity in sea urchin eggs was identical to CK2.     

Phylogeny of regulatory proteins of the complement system. Isolation and characterization of a C4b/C3b inhibitor and a cofactor from sand bass plasma

We have previously demonstrated that the alpha'-chain of human activated form of the fourth (C4b) and third (C3b) component of C are cleaved by plasma or serum from vertebrate species spanning through 300,000,000 yr of evolution yielding fragments identical with those obtained with human plasma. In this study, we investigated the molecular basis of this reaction. We chose barred sand bass plasma because this is the most primitive species analyzed possessing these activities. Barred sand bass plasma proteins were separated on a Sephadex G-200 column and the eluted samples analyzed for C4b and C3b cleavage. Individual fractions were inactive, but degradation was obtained when proteins of 380 and 155 kDa were combined. In contrast to the human regulatory proteins, the sand bass proteins require Ca2+ ions. K76COOH, an inhibitor of human factor I, inhibited the function of the 155-kDa but not of the 380 kDa-fraction. Thus it appears that the 155-kDa fraction functions as the C4b/C3b cleaving enzyme (I) and the 380-kDa material as its cofactor. Further purification of the 380-kDa fraction yielded a protein that by SDS-PAGE consisted of two noncovalently linked subunits of 110 and 42 kDa at a molecular ratio of 2:1. These two chains were antigenically distinct, and constitute domains of the same protein. The 110-kDa peptide binds C4b and not C3b but it fully expresses the cofactor function for the 155-kDa fraction on the cleavage of both C4b and C3b. Limited tryptic digestion of the 110-kDa domain demonstrated C4b binding activity in fragments of 34, 25, and 23 kDa. The activity of the 34-kDa fragment was the same as that of the undigested protein. Comparison of the amino acid composition of the barred sand bass cofactor and of human C4bp shows similar high content of cysteine and proline but not of tryptophan. It differs from human factor H in cysteine, serine, proline, and tryptophan. These studies indicate that regulatory proteins for the C4b and C3b C fragments may have appeared very early phylogenetically.     

Immunological Identification of Multiple α-Like Subunits of the γ-Aminobutyric AcidA Receptor Complex Purified from Neonatal Rat Cortex

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.     

Partial purification and characterization of erythropoietin receptors from erythroid progenitor cells

We have partially purified and characterized erythropoietin (Epo) receptors of erythroid progenitor cells which were obtained from the spleens of anemia-inducing Friend virus infected mice. Membrane proteins of splenic erythroid progenitor cells were solubilized with 1% Triton X-102. Upon chromatography on DEAE-Sephacel anion-exchange columns, two distinct Epo receptor peak fractions referred to as Peak I and Peak II were identified by 125I-Epo binding assays using the polyethylene glycol precipitation method. The Peak I and Peak II samples were then individually chromatographed on an S-Sepharose column. The S-Sepharose-purified Peak I and Peak II samples were crosslinked with 125I-Epo in the presence and absence of excess unlabeled Epo by disuccinimidyl suberate treatment, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Both Peak I and Peak II samples showed a radiolabeled peptide with a Mr 135K and the labeling was blocked by excess unlabeled Epo. Since the Mr of Epo is about 35K, Epo receptor peptide has a Mr approximately 100K. To determine whether Epo stimulates autophosphorylation of the receptors, the S-Sepharose-purified Peak I and Peak II samples were incubated with or without Epo, and then briefly incubated in the presence of [gamma-32P]ATP and Mn2+. The tyrosine residue phosphorylated protein was isolated by an immunochemical technique, and then analyzed by SDS-PAGE and autoradiography. The result showed that Epo stimulates phosphorylation of a 100-kDa peptide.     

A domain of synapsin I involved with actin bundling shares immunologic cross-reactivity with villin

Synapsin I is a neuronal phosphoprotein that can bundle actin filaments in vitro. This activity is under phosphorylation control, and may be related to its putative in vivo role of regulating the clustering and release of small synaptic vesicles. We have compared human and bovine synapsin I by peptide mapping, and have used NTCB (2-nitro-5-thiocyano benzoic acid) cleavage to generate a series of peptide fragments from bovine synapsin I. After chymotryptic digestion, 88% of the tyrosine-containing fragments appear to be structurally identical in human and bovine synapsin I, as judged by their positions on high-resolution two-dimensional peptide maps. The alignment of the NTCB peptides within the parent protein have been determined by peptide mapping, and the ability of these fragments to precipitate with actin bundles has been measured. Only peptides that are derived from regions near the ends of the protein are active. One such 25-kDa peptide which sediments with actin also cross-reacts with antibodies to chicken villin, an actin binding and bundling protein derived from the intestinal microvillus. Since in other respects villin appears to be an unrelated protein, these results suggest the possibility that certain actin binding proteins may show immunologic cross-reactivity due to convergent evolution within the acting binding domain.     

Expression of variant forms of proopiomelanocortin, the common precursor to corticotropin and beta-lipotropin in the rat pars intermedia

Proopiomelanocortin, the common glycoprotein precursor to adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), is the most abundant protein synthesized in rat neurointermediate lobes. It represents 30% of the total amount of radioactive proteins obtained after a 1-h pulse incubation with [3H]phenylalanine. Several forms of this protein can be separated by a high-resolution two-dimensional gel electrophoresis technique. The three most abundant species which can be reproducibly characterized by their apparent molecular weights (Mr) and isoelectric points (pI) were called form I (Mr 34 000; pI 8.2), form II (Mr 36 000; pI 8.2), and form III (Mr 35 000; pI 7.3). Additional minor forms, representing together approximately 30% of the total forms I, II, and III combined, are also observed. They have very close molecular weights but differ by their isoelectric points. When glycosylation is prevented by tunicamycin, forms I and II are replaced by a new molecule with the same pI of 8.2 but a slightly lower Mr (32 000). This form is referred to as form T1. Similarly, form III is replaced by form T2 (Mr 33 000; pI 7.3). Forms T1 and T2 are supposed to be nonglycoslyated peptides. They were further characterized by microsequencing and peptide mapping. They both have the same N-terminal amino acid sequence with leucine residues in positions 3 and 11, and they both contain identical [3H]phenylalanine-labeled tryptic fragments, two of them corresponding to the sequences 1-8 of ACTH and 61-69 of beta-LPH. However, a limited digestion with the Staphylococcus aureus (V8 strain) protease generates a collection of peptides different for each form. These results suggest the presence of at least two different gene products corresponding to the major forms of proopiomelanocortin in the rat pars intermedia.     

Insulin and 12-O-tetradecanoylphorbol-13-acetate activation of two immunologically distinct myelin basic protein/microtubule-associated protein 2 (MBP/MAP2) kinases via de novo phosphorylation of threonine and tyrosine residues.

Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.     

Stimulation of the phosphorylation of cytoskeletal 350-kDa and 300-kDa proteins by insulin-like growth factor-I, platelet-derived growth factor and phorbol ester in rat 3Y1 cells

Insulin-like growth factor-I (IGF-I) stimulated the phosphorylation of cytoskeletal 350-kDa and 300-kDa proteins which were immunoprecipitated with antibodies against brain high molecular weight microtubule-associated proteins in quiescent rat 3Y1 cells. The data on the effective concentrations of IGF-I and 125I-labeled IGF-I binding indicated that type I IGF receptors mediate this IGF-I effect. Platelet-derived growth factor (PDGF) as well as phorbol ester (TPA) also stimulated the phosphorylation of these proteins. These proteins, whether immunoprecipitated from cells stimulated by insulin, IGF-I, TPA, PDGF, or epidermal growth factor, produced very similar phosphopeptide mapping patterns irrespective of the stimulant. The results suggest the possibility that these growth factors and phorbol esters may activate a common protein kinase which is responsible for the phosphorylation of the 350-kDa and 300-kDa proteins in cells.     

Characterization of a novel isozyme of cGMP-dependent protein kinase from bovine aorta

Two isozymic forms of cGMP-dependent protein kinase (designated types I alpha and I beta) were purified to homogeneity from bovine aorta smooth muscle. Type I alpha was apparently the same as the well characterized bovine lung cGMP-dependent protein kinase. Type I beta had a subunit Mr = 80,000 compared with Mr = 78,000 for type I alpha, and both forms were dimeric with similar calculated native Mr (170,000-178,000). Both enzymes contained two cGMP-binding sites per subunit, exhibited similar specificities for the peptide substrates tested, photoaffinity labeled with 8-N3[32P] cAMP, and catalyzed autophosphorylation. Silver-stained peptide maps of types I alpha and I beta were similar but not identical; however, autoradiographs of peptide maps of these enzymes prelabeled by either autophosphorylation or photoaffinity labeling showed clearly different patterns. The amino-terminal sequence of a breakdown product of type I beta could not be aligned confidently with any of the published sequence of bovine lung cGMP-dependent protein kinase. [3H]cGMP dissociation curves for types I alpha and I beta were both biphasic, but the dissociation rate of the slow component of type I beta was faster than the corresponding component of type I alpha. The concentration of cGMP required for half-maximal activation (K alpha) was slightly lower for type I alpha than for type I beta (0.29 and 0.44 microM, respectively), and the two enzymes had similar K alpha values for cAMP (16 and 18 microM, respectively). Types I alpha and I beta exhibited different K alpha values for several cGMP analogs. The abundance of type I beta in specific tissues suggested that it could have an important physiological role.     

Identification of integrin-like matrix receptors with affinity for interstitial collagens

Antibodies to a rat liver membrane glycoprotein with an Mr of 115,000 (nonreduced) inhibited the attachment of rat hepatocytes and primary rat heart fibroblasts to both collagen and fibronectin. The Mr 115,000 glycoprotein cross-reacted immunologically with the beta 1-chain of the rat hepatocyte fibronectin receptor (HFNR), and the two proteins showed identical peptide maps after proteolytic cleavage. It was concluded that the Mr 115,000 protein was similar or identical to the beta 1-chain of Arg-Gly-Asp (RGD)-directed matrix receptors. Although collagen type I contains several RGD sequences, the attachment of hepatocytes and fibroblasts to collagen type I was not inhibited by the synthetic peptide GRGDTP in concentrations that blocked adhesion to fibronectin. Furthermore, hepatocytes adhered equally well to collagen fragments, generated by cyanogen bromide cleavage, lacking RGD sequences as to fragments containing this sequence. Antibodies to the Mr 115,000 protein inhibited the adhesion of hepatocytes to both types of collagen fragments. Taken together, these data indicate the presence of collagen receptors that share the beta-subunit with the HFNR but that are not directed to RGD sequences. Tentative alpha-chains of the collagen matrix receptor complex were isolated by immunoprecipitation of surface 125I-labeled fibroblast membrane proteins purified by affinity chromatography on immobilized collagen type I. Data are presented indicating that proteins with Mr around 145,000 and 170,000 (nonreduced) are associated in noncovalently linked complexes with the Mr 115,000 protein. These complexes have affinity for collagen and thus have properties expected for integrin-like collagen receptors.