Identification in rat brain of a 19-kDa protein that comigrates on two-dimensional electrophoresis with p19, a hormonally regulated phosphoprotein of insulinoma cells

We have previously shown that a set of 19-kDa cytosolic proteins, p19, undergoes hormone-dependent phosphorylation in several peptide hormone-producing tumor cells. Here we show, using comigration on two-dimensional electrophoresis with RIN-1122 rat insulinoma cell p19, that an identical set of 19-kDa proteins is present in rat brain but not in liver or skeletal muscle. We have partially purified p19 from rat brain and have compared the apparent isoelectric variants by tryptic peptide mapping. The data suggest that p19 is a novel phosphoprotein consisting of an unphosphorylated form and of three phosphoforms.     

Selective phosphorylation of the alpha subunit of the sodium channel by cAMP-dependent protein kinase

The alpha subunit of the sodium channel purified from rat brain is rapidly and selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 3 to 4 mol of 32P/mol of saxitoxin-binding activity. The rate of phosphorylation is comparable to that of the synthetic peptide analog of the phosphorylation site of pyruvate kinase, one of the best substrates for cAMP-dependent protein kinase. An endogenous cAMP-dependent protein kinase that is present in the partially purified sodium channel preparations also selectively phosphorylates the alpha subunit. The specificity and rapidity of the phosphorylation reaction are consistent with the hypothesis that the alpha subunit is phosphorylated by cAMP-dependent protein kinase in vivo.     

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.     

Regulatory subunit of the type I cAMP-dependent protein kinase as an inhibitor and substrate of the cGMP-dependent protein kinase

The regulatory subunit of the type I cAMP-dependent protein kinase (Rt) serves as a substrate for the phosphotransferase reaction catalyzed by cGMP-dependent protein kinase (Km = 2.2 microM). The reaction is stimulated by cGMP when RI . cAMP is the substrate, but not when nucleotide-free RI is used. The cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of RI dimer in the presence of cAMP and a self-phosphorylation reaction to the extent of 4 mol of phosphate/mol of enzyme dimer. In the absence of cAMP, RI is a competitive inhibitor of the phosphorylation of histone H2B (Ki = 0.25 microM) and of the synthetic peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ki = 0.15 microM) by the cGMP-dependent enzyme. Nucleotide-free RI also inhibits the intramolecular self-phosphorylation of cGMP-dependent protein kinase. The inhibition of the phosphorylation reactions are reversed by cAMP. The catalytic subunit of cAMP-dependent protein kinase does not catalyze the phosphorylation of RIand does not significantly alter the ability of RI to serve as a substrate or an inhibitor of cGMP-dependent protein kinase. These observations are consistent with the concept that the cGMP- and cAMP-dependent protein kinases are closely related proteins whose functional domains may interact.     

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.     

Phosphorylation of tubulin by a calmodulin-dependent protein kinase

Calmodulin-dependent protein kinase was purified from porcine brain cytosol through sequential steps involving acid precipitation, DEAE-chromatography, and calmodulin-Sepharose chromatography. The purified enzyme contained a major Mr 50,000 and a minor Mr 60,000 peptide. Porcine brain tubulin was a major substrate for this kinase. Under optimal conditions 2.6 mol of phosphate were incorporated per mol of tubulin. The kinase phosphorylated both tubulin subunits at their carboxyl-terminal region. Limited proteolysis, using trypsin and chymotrypsin, of phosphorylated and unphosphorylated tubulins resulted in different cleavage patterns as determined by peptide mapping. Phosphorylated tubulin was unable to bind to microtubule-associated protein or to polymerize, but regained its assembly capacity after phosphatase treatment.     

Tyrosine kinase catalyzed phosphorylation and inactivation of the inhibitor protein of the cAMP-dependent protein kinase

The inhibitor protein of the cAMP-dependent protein kinase is a potential high affinity regulator of cAMP function. We now show that it is phosphorylated in Tyr7 by the intrinsic tyrosine kinase activity of epidermal growth factor receptor. The phosphorylated form can be readily separated from the unphosphorylated protein by high pressure liquid chromatography which has permitted the isolation of stoichiometrically phosphorylated protein. Using this method, it has been demonstrated that this phosphorylation, which occurs within the inhibitor protein's active domain, results in a 6 to 9-fold decrease in inhibitory potency. Possibly, a component of growth control could be the coupling of tyrosine kinase activity to cAMP-mediated cellular proliferation via the regulation of the efficacy of the inhibitor protein.     

Microtubule-associated protein tau is phosphorylated by protein kinase C on its tubulin binding domain.

We have analyzed the in vitro phosphorylation of tau protein by Ca2+/calmodulin-dependent protein kinase, casein kinase II, and proline-directed serine/threonine protein kinase. These kinases phosphorylate tau protein in sites localized in different regions of the molecule, as determined by peptide mapping analyses. Focusing on the phosphorylation of tau by protein kinase C, it was calculated as an incorporation of 4 mol of phosphate/mol of tau. Limited proteolysis assays suggest that the phosphorylation sites could be located within the tubulin-binding domain. Direct phosphorylation of synthetic peptides corresponding to the cysteine-containing tubulin-binding region present in both fetal and adult tau isoforms demonstrates that serine 313 is modified by protein kinase C. Phosphorylation of the synthetic peptide by protein kinase C diminishes its binding to tubulin, as compared with the unphosphorylated peptide.     

cAMP-dependent phosphorylation and hexamethylene-bis-acetamide induced dephosphorylation of p19 in murine erythroleukemia cells

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP- dependent kinase creates the more acidic species.     

Inhibitors of protein phosphatase-1. Inhibitor-1 of bovine adipose tissue and a dopamine- and cAMP-regulated phosphoprotein of bovine brain are identical

Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-1 was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase

Protein kinase C incorporates phosphate into two sites of myosin light chain kinase (MLC-kinase) in the absence of calmodulin. Phosphorylation is all but abolished in the presence of Ca2+ and calmodulin, suggesting that both sites of phosphorylation are close to the calmodulin binding site. The phosphorylation of MLC-kinase results in an approximately 10-fold increase in the dissociation constant of MLC-kinase for calmodulin. Following phosphorylation (2 mol/mol of enzyme) of MLC-kinase by protein kinase C, an additional 2 mol of phosphate can be incorporated into the MLC-kinase apoenzyme by the cAMP-dependent protein kinase. Different maps of phosphopeptides were obtained by tryptic hydrolysis from MLC-kinase preparations phosphorylated by each kinase. The phosphorylation sites for the cAMP-dependent kinase were located in a fragment of approximately 25,000 daltons. In contrast the phosphorylation sites for protein kinase C are found in a much smaller tryptic peptide. These results suggest that the phosphorylation sites on MLC-kinase are different for protein kinase C and for cAMP-dependent protein kinase. However, phosphorylation in both regions results in a reduced affinity for calmodulin.     

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.     

Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase

Rabbit myelin basic protein (MBP) was phosphorylated by a ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/mol MBP in the presence and absence of GTlb, respectively. Two-dimensional peptide mapping analyses revealed that two of the sites of phosphorylation were distinct from those catalyzed by cAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by GTlb, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.     

Expression of phosphoprotein p19 in brain, testis, and neuroendocrine tumor cells. Developmental regulation in rat brain

We have recently purified from bovine brain a 19-kDa protein, p19, that was previously shown to undergo hormonally regulated phosphorylation in several neuroendocrine tumor cells. We now report the tissue distribution of p19, studied by immunoblotting. Using a rabbit antiserum, which binds both to the unphosphorylated form and to the two predominant phosphoforms of p19, we show that the protein is present in brain and testis but not in a variety of other mammalian tissues. High levels of p19 are also present in several cultured tumor cells expressing neuroendocrine properties. In addition, p19 was detected in HL60 promyelocytic leukemia and in Friend erythroleukemia cells, but not in several other cell lines. In rat brain, we show that the level of p19 is maximal on the first postnatal day and declines within the first 2 weeks of life to a low plateau that persists into adulthood. The concentration of translatable p19 mRNA also decreases postnatally in rat brain, suggesting that the developmental regulation of the expression of p19 occurs, at least in part, at a pretranslational level. The broad species cross-reactivity of the p19 antibody suggests that the gene encoding p19 has been highly conserved during mammalian evolution. Based on the pattern of expression of this protein, we propose that p19 plays a role in the development of neurons and neuroendocrine cell types.     

Purification and characterization of a 22-kDa microsomal protein from rat parotid gland which is phosphorylated following stimulation by agonists involving cAMP as second messenger

Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 (protein I) and two other particulate proteins with apparent molecular masses of 24 kDa (protein II) and 22 kDa (protein III) [Jahn, R., Unger, C. & S?ling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. This report describes the purification and characterization of protein III. Solubilization studies indicate that protein III is an intrinsic membrane protein. It could be extracted from the endoplasmic reticulum membrane only with Triton X-100, SDS or concentrated formic or acetic acid. The purification of this protein involved extraction of the microsomes with Triton X-100, removal of the detergent by acetone precipitation, extraction of water-soluble proteins, lipids and lipoproteins, and preparative SDS polyacrylamide gel electrophoresis. The protein has a basic pI (greater than 8.7). For determination of the amino acid composition of protein III and for sequencing of its amino-terminal portion, the protein was electroeluted out off the gel, the detergent removed and the protein finally purified by reversed-phase HPLC. Protein III could be phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase to a degree of approximately 0.14 mol phosphate/mol protein. The only phosphopeptide obtained after in vitro phosphorylation and subsequent tryptic or chymotryptic digestion was identical with the phosphopeptide obtained after stimulation of intact rat parotid gland lobules with isoproterenol. The sequence of this peptide was Lys-Leu-Ser(P)-Glu-Ala-Asp-Asn-Arg. It was confirmed by an analysis of the synthetic peptide following in vitro phosphorylation with cAMP-dependent protein kinase. The first 41 N-terminal residues of protein III were sequenced. So far no sequence homology with other known peptides or proteins could be found.     

Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.     

Regulation of NAD-dependent glutamate dehydrogenase by protein kinases in Saccharomyces cerevisiae

The active NAD-dependent glutamate dehydrogenase of wild type yeast cells fractionated by DEAE-Sephacel chromatography was inactivated in vitro by the addition of either the cAMP-dependent or cAMP-independent protein kinases obtained from wild type cells. cAMP-dependent inhibition of glutamate dehydrogenase activity was not observed in the crude extract of bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase. The cAMP-dependent protein kinase of CYR3 mutant cells, which has a high K alpha value for cAMP in the phosphorylation reaction, required a high cAMP concentration for the inactivation of NAD-dependent glutamate dehydrogenase. An increased inactivation of partially purified active NAD-dependent glutamate dehydrogenase (Mr = 450,000) was observed to correlate with increased phosphorylation of a protein subunit (Mr = 100,000) of glutamate dehydrogenase. The phosphorylated protein was labeled by an NADH analog, 5'-p-fluorosulfonyl[14C]benzoyladenosine. Activation and dephosphorylation of inactive NAD-dependent glutamate dehydrogenase fractions were observed in vitro by treatment with bovine alkaline phosphatase or crude yeast cell extracts. These results suggested that the conversion of the active form of NAD-dependent glutamate dehydrogenase to an inactive form is regulated by phosphorylation through cAMP-dependent and cAMP-independent protein kinases.     

Modulation of red cell band 4.1 function by cAMP-dependent kinase and protein kinase C phosphorylation

Human erythrocyte protein 4.1 is phosphorylated in vivo by several protein kinases including protein kinase C and cAMP-dependent kinase. We have used cAMP-dependent kinase purified from red cells and protein kinase C purified from brain to test the effects of phosphorylation on band 4.1 function. In solution, each kinase catalyzed the incorporation of 1-4 mol of PO4/mol of band 4.1. Phosphorylation of band 4.1 by each kinase resulted in a significant (50-80%) reduction in the ability of band 4.1 to promote spectrin binding to F-actin. Direct measurement of spectrin-band 4.1 binding showed that phosphorylation by each kinase also caused dramatic reduction in this association. Phosphorylation of band 4.1 by each kinase for increasing time periods enabled us to demonstrate an approximately linear inverse relationship between PO4 incorporation into band 4.1 and spectrin binding. These results show that phosphorylation of band 4.1 by cAMP-dependent kinase and protein kinase C may be central to the regulation of red cell cytoskeletal organization and membrane mechanical properties.     

Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.