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At least 19 recordsLinked to original sources
PubMed1988

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.

Amino Acid Sequence↗
PubMed1986

Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus.

We provide direct evidence that serine 17 is the major site of serine phosphorylation in p60v-src, the transforming protein of Rous sarcoma virus, and in its cellular homolog, p60c-src. The amino acid composition of the tryptic peptide containing the major site of serine phosphorylation in p60v-src was deduced by peptide map analysis of the protein labeled biosynthetically with a variety of radioactive amino acids. Manual Edman degradation revealed that the phosphorylated serine in this peptide was the amino terminal residue. These data are consistent only with the phosphorylation of serine 17. The major site of serine phosphorylation in chicken p60c-src, the cellular homolog of p60v-src, is contained in a tryptic peptide identical to that containing serine 17 in p60v-src of Schmidt Ruppin Rous sarcoma virus of subgroup A. Serine 17 is therefore also phosphorylated in p60c-src. The p60v-src protein encoded by Prague Rous sarcoma virus was found to contain two sites of tyrosine phosphorylation. The previously unrecognized site of tyrosine phosphorylation may be tyrosine 205 or possibly tyrosine 208. Treatment of Prague Rous sarcoma virus-infected cells with vanadyl ions stimulated the protein kinase activity of p60v-src and increased the phosphorylation of tyrosine 416 but not the phosphorylation of the additional site of tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed1987

Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation.

Smooth muscle heavy meromyosin (HMM) is phosphorylated by the Ca2+-activated phospholipid-dependent protein kinase, i.e. protein kinase C, at three sites on each 20,000-dalton light chain. Phosphorylation of three sites also is observed with isolated 20,000-dalton light chain and HMM subfragment 1. The phosphorylation sites are serine 1, serine 2, and threonine 9. Threonine is phosphorylated most rapidly followed by either serine 1 or 2. Phosphorylation of the third site occurs only on prolonged incubation. Phosphorylation is a random process. HMM phosphorylated at two sites per light chain by protein kinase C can be dephosphorylated, as shown using two phosphatase preparations. Increasing levels of phosphorylation of HMM by protein kinase C causes a progressive inhibition of the subsequent rate of phosphorylation of serine 19 by myosin light chain kinase and causes a progressive inhibition of actin-activated ATPase activity of HMM, prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of HMM for actin rather than a change in Vmax. Previous results with HMM and protein kinase C (Nishikawa, M., Sellers, J. R., Adelstein, R. S., and Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814) examined effects induced by phosphorylation of the threonine residues. Our results confirm these and consider also the influence of higher levels of phosphorylation by protein kinase C.

Amino Acid Sequence↗
PubMed1986

Studies on the methods for the determination of phosphorylation sites in highly phosphorylated peptides or proteins: phosphorylation sites of hen egg white riboflavin binding protein.

To determine the phosphate binding sites in hen egg white riboflavin binding protein (RBP), a highly phosphorylated peptide, which consisted of 23 amino acid residues including eight phosphoserines, was isolated from the tryptic digest of reduced and carboxymethylated RBP. The conditions of the beta-elimination-addition reaction to convert phosphoserine residues in the peptide to cysteic acids, S-methylcysteines, alanines, and beta-methylaminoalanines (DL-alpha-amino-beta-methylamino propionic acid) were examined. These converted peptides were purified by HPLC and subjected to Edman degradation. The results of Edman degradation indicated that the S-methylcysteine derivative of the peptide gave the most satisfactory result for determining the phosphate binding sites in the peptide. The phosphorylation sites of the peptide determined by the method mentioned above are as follows: His182-Leu-Leu-Ser185-Glu-Ser(P)-Ser(P)-Glu-Glu190-Ser (P)-Ser(P)-Ser(P)-Met-Ser195(P)-Ser(P)-Ser(P)-Glu-Glu-. These studies indicated that the conversion of phosphoserines in phosphoproteins to S-methylcysteines followed by Edman analysis was a useful method for the elucidation of the phosphorylation sites in phosphopeptides.

Amino Acid Sequence↗
PubMed1984

Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP.

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).

Amino Acids↗
PubMed1986

Phosphorylation of tyrosine hydroxylase on at least three sites in rat pheochromocytoma PC12 cells treated with 56 mM K+: determination of the sites on tyrosine hydroxylase phosphorylated by cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases.

Incubation of rat pheochromocytoma PC12 cells with the calcium ionophore, A23187 (10(-5) M), 56 mM K+, or dibutyryl cAMP (2 mM) is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in the cells. Both the activation and the increased phosphorylation of tyrosine hydroxylase produced by A23187 and 56 mM K+ are dependent on the presence of extracellular calcium, whereas similar effects produced by dibutyryl cAMP are independent of calcium. The effects of 56 mM K+ plus dibutyryl cAMP or A23187 plus dibutyryl cAMP on the activation and phosphorylation of tyrosine hydroxylase are additive. In contrast, the effects of 56 mM K+ plus A23187 on either the activation or the phosphorylation of the enzyme are not additive. Following stimulation of intact PC12 cells with 32Pi, in order to label ATP stores, and tryptic digestion of the phosphorylated enzyme, separation of the tryptic phosphopeptides by high pressure liquid chromatography yields four distinct 32P-peptide peaks. Incubation of the cells in the presence of either 56 mM K+ or A23187 is associated with increased 32Pi incorporation into three peptides whereas, in the presence of dibutyryl cAMP, increased 32Pi incorporation is observed in only one of these peptides. When tyrosine hydroxylase purified from rat pheochromocytoma tumor is incubated in vitro with [gamma-32P]ATP and either cAMP-dependent or calcium/calmodulin-dependent protein kinase under appropriate conditions, increased phosphorylation of tyrosine hydroxylase is observed. However, even though in vitro phosphorylation by cAMP-dependent protein kinase is associated with activation of tyrosine hydroxylase, in vitro phosphorylation by calcium/calmodulin-dependent protein kinase does not lead to activation of the enzyme. Tryptic digestion of tyrosine hydroxylase phosphorylated by calcium/calmodulin-dependent protein kinase yields three distinct 32P-peptide peaks, which are identical to those phosphorylated by treatment of intact PC12 cells with either high K+ or A23187. In contrast, cAMP-dependent protein kinase phosphorylates only one peptide, which is identical to that phosphorylated by treatment of the intact cells with dibutyryl cAMP. These results indicate that tyrosine hydroxylase is activated and phosphorylated at multiple sites in PC12 cells exposed to 56 mM K+ or A23187. The results suggests that the in situ phosphorylation of these sites is catalyzed by calcium/calmodulin-dependent protein kinase; however, phosphorylation by this protein kinase is not sufficient to activate the enzyme.

Adrenal Gland Neoplasms↗
PubMed1991

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.

Amino Acids↗
PubMed1989

Spatial and temporal expression of phosphorylated and non-phosphorylated forms of neurofilament proteins in the developing nervous system of Xenopus laevis.

Immunocytochemical studies of developing Xenopus laevis embryos and tadpoles (stages 12 1/2 to 46) were performed using a panel of 11 monoclonal antibodies to phosphorylated and non-phosphorylated forms of the neurofilament proteins. These included nine antibodies to the middle molecular weight neurofilament protein (XNF-M, 175 kDa), and two additional antibodies to non-phosphorylated forms of the other two neurofilament proteins (XNF-L, 73 kDa; XNF-H, 205 kDa). The developmental expression of XNF-M, XNF-L and XNF-H, and the progressive phosphorylation of XNF-M in the rhombencephalon, spinal cord, and optic nerve were studied using these antibodies. In the spinal cord and rhombencephalon, non-phosphorylated forms of XNF-M were initially detected during neural tube stages (stages 22-26), one day before XNF-L and XNF-H at early tadpole stages (stage 35/36). In the eye, XNF-M was observed initially during tailbud stages (stage 29/30), but neither XNF-L nor XNF-H was seen even by stage 46 (swimming tadpole). The phosphorylation of XNF-M occurred over a protracted period of several days, both in the neural tube and visual system, and could be divided into four phases. (1) When initially expressed, XNF-M was hypophosphorylated. This was indicated by the early immunostaining of axons and cell bodies with antibodies to dephosphorylated epitopes on XNF-M and by the absence of staining with antibodies to phosphorylated epitopes. (2) After a short timelag (3-9 h) axons were stained by some, but not all antibodies to phosphorylated epitopes. (3) Approximately one day later, all antibodies to phosphorylated epitopes stained the relevant axons. However, XNF-M was not yet fully phosphorylated, as indicated by the continued staining of these axons with antibodies to dephosphorylated epitopes of XNF-M. (4) Two to 3 days after the initial expression of XNF-M, dephosphorylated epitopes disappeared from the axons, establishing the adult pattern. During development, the most heavily phosphorylated neurofilament proteins present at a given stage were found first in distal regions of the axons and progressed gradually toward the neuronal perikarya as development proceeded. This gradient of phosphorylation, established early within the axon, suggests that neurofilaments in the axons mature from their distal ends toward the cell body, a process which may be regulated by local factors within the axons themselves. The similarity of the basic features of NF-M phosphorylation in mammalian, avian, and amphibian axons underscores the importance of this phenomenon for the development of a mature axon.

Aging↗
PubMed1989

Effect of insulin on ATP-citrate lyase phosphorylation: regulation of peptide A and peptide B phosphorylations.

Insulin decreases multifunctional protein kinase (MFPK) activity in rat adipose tissue [Ramakrishna, S., & Benjamin, W. B. (1988) J. Biol. Chem. 263, 12677-12681]. Insulin also decreases the phosphorylation of peptide B but increases the phosphorylation of peptide A of ATP-citrate lyase (ATP-CL). The mechanism for this increase in peptide A phosphorylation was studied with purified ATP-CL from control and insulin- and isoproterenol-treated fat pads by using MFPK and the catalytic subunit of cAMP-dependent protein kinase (A-kinase). ATP-CL purified from insulin-treated fat pads is a better substrate for phosphorylation by MFPK compared to controls. This result is consistent with the hypothesis that insulin action decreases peptide B phosphorylation. To determine if the degree of phosphorylation at peptide B affects the phosphorylation rate of peptide A by A-kinase, ATP-CL was prepared with determined phosphate contents of peptides A and B. ATP-CL with a low phosphate content at peptide B is a better substrate for phosphorylation at peptide A by A-kinase than is ATP-CL with a high phosphate content at peptide B. These results suggest that the insulin-induced increase in ATP-CL phosphorylation at peptide A is due to a decrease in peptide B phosphorylation. ATP-CL prepared from isoproterenol-treated fat pads is also a better substrate for phosphorylation at peptide B by MFPK than controls. This increase in phosphorylation at peptide B by MFPK is due to positive second-site regulation by the isoproterenol-induced increase in peptide A phosphorylation.

ATP Citrate (pro-S)-Lyase↗
PubMed1988

Phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase. Temporal correlation of acetylcholine's effect on site phosphorylation, enzyme activation, and catecholamine synthesis.

Tryptic peptide fragments of tyrosine hydroxylase isolated from 32PO4-prelabeled bovine adrenal chromaffin cells are resolved into seven phosphopeptides by reverse phase-high performance liquid chromatography. All seven of the peptides are phosphorylated on serine residues. Three of these putative phosphorylation sites, peptides 3, 5, and 6, are rapidly phosphorylated (5-fold in 15 s) by both acetylcholine stimulation and potassium depolarization of the cells, and this phosphorylation is accompanied by a similarly rapid activation of the enzyme. Both phosphorylation and activation are transient and do not account for the prolonged increase in catecholamine biosynthesis produced by these stimuli. Peptides 4 and 7 show a much slower and sustained increase in phosphorylation (3-fold in 4 min) in response to acetylcholine and potassium. Phosphorylation of these peptides correlates with the sustained increase in catecholamine biosynthesis rather than enzyme activation. Peptides 1 and 2 are not stimulated by any agonist yet employed and thus show no relation to enzyme activation or catecholamine biosynthesis. Phosphorylation of all five peptides by acetylcholine or potassium is calcium-dependent. In contrast to the stimulation of phosphorylation of tyrosine hydroxylase on multiple sites, forskolin stimulates the phosphorylation of only peptide 6, and this is accompanied by a coordinated activation of tyrosine hydroxylase and increased catecholamine biosynthesis. These findings show that the phosphorylation of tyrosine hydroxylase in intact cells is more complex than predicted from in vitro results, that at least two protein kinases are involved in the secretagogue-induced phosphorylation of tyrosine hydroxylase, and that the regulation of catecholamine biosynthesis, in response to phosphorylation, appears to involve both tyrosine hydroxylase activation and other mechanisms.

Acetylcholine↗
PubMed1986

A simple and rapid preparation of fully phosphorylated and fully dephosphorylated skeletal muscle myosin. Application to the preparation of a phosphorylated LC2-modified artificial isozyme.

Fast skeletal myosin LC2 is phosphorylated on ser-15 by a specific myosin light chain kinase (MLCK) in the presence of Ca2+ and calmodulin, and dephosphorylated by a muscle phosphate in the presence of Mg2+. Fully dephosphorylated myosin is obtained by dialysis of muscle crude extract (0.06 M NaCl, 0.01 M Tris-HCl, pH 7.5, 50 microM EGTA); fully phosphorylated myosin is obtained by addition of Ca2+ (0.2 mM), Mg2+ (10 mM) and ATP (3 mM) and 5 min incubation at 28 degrees C. The following reaction characteristics were noted. The crude extract is a very efficient phosphorylating complex and can be diluted to phosphorylate or dephosphorylate purified myosin. Phosphorylation and dephosphorylation appear monophasic, showing no evidence of negative cooperativity in this particular type of myosin and medium. Phosphorylation is 24 times slower in the presence of 0.45 M KCl, 5 mM pyrophosphate. Thiophosphorylated myosin is slowly dephosphorylated by phosphatase. At the crude myosin stage the dephosphorylation reaction is efficiently inhibited (at 0-4 degrees C) by the presence of 70 mM NaF. Myosin-[(T)-LC2'] (a myosin species in which LC2 has been selectively modified by trypsin) is an interesting species refractory to phosphorylation. The myosin-[(T)-LC2'] isozyme can be obtained fully phosphorylated by phosphorylation of myosin followed by limited tryptic proteolysis as described earlier. Urea-PAGE as used separates LC2, phosphoryl-LC2, LC2' and phosphoryl-LC2' effectively and in this order. Through this procedure the (de)-phosphorylating complex is ipso facto specific to the myosin species considered; the method avoids lengthy preparations of purified proteins and is easy, rapid and efficient.

Animals↗
PubMed1988

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components.

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.

Acetone↗
PubMed1990

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II: comparative study of the phosphorylation sites.

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 +/- 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 +/- 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 +/- 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca2+/CaM-binding.

Amino Acid Sequence↗
PubMed1985

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation.

Isolated microtubule-associated protein 2 (MAP2), tau factor, and tubulin were phosphorylated by a purified Ca2+, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and tau factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, tau factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The Km values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, tau factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca2+, calmodulin system via phosphorylation of MAP2, tau factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.

Animals↗
PubMed1990

Phosphorylation of charge isomers (components) of human myelin basic protein: identification of phosphorylated sites.

Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM-52 column chromatography. Two of the components C-1 and C-4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N-terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5-13 was approximately 10 times greater than that of any of the other peptides derived from C-1, while the rate of phosphorylation of peptide 5-13 derived from C-4 was 10-20 times greater than that of any of the other peptides derived from C-4. In addition, peptide 5-13, which contained a major phosphorylation site in both C-1 and C-4, was phosphorylated at a faster rate in C-4 (460 cpm/nM/min) compared with C-1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of beta-structure in a previous study. In that study, C-1, which contained about 13% beta-structure before phosphorylation, increased to approximately 40% after phosphorylation. Construction of a model peptide of this N-terminal region, which included the phosphorylation site at Ser7, demonstrated that the beta-structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidyl groups of Arg5 and Arg9.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1991

Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin.

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.

Amino Acid Sequence↗
PubMed1989

Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation.

Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. In cell-free systems, cAMP-dependent protein kinase catalyzes exclusive phosphorylation of serine 16 of phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives exclusive phosphorylation of threonine 17 (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). In this work we have localized the sites of phospholamban phosphorylation in intact ventricles treated with the beta-adrenergic agonist isoproterenol. Isolation of phosphorylated phospholamban from 32P-perfused guinea pig ventricles, followed by partial acid hydrolysis and phosphoamino acid analysis, revealed phosphorylation of both serine and threonine residues. At steady state after isoproterenol exposure, phospholamban contained approximately equimolar amounts of these two phosphoamino acids. Two major tryptic phosphopeptides containing greater than 90% of the incorporated radioactivity were obtained from phospholamban labeled in intact ventricles. The amino acid sequences of these two tryptic peptides corresponded exactly to residues 14-25 and 15-25 of canine cardiac phospholamban, thus localizing the sites of in situ phosphorylation to serine 16 and threonine 17. Phosphorylation of phospholamban at two sites in heart perfused with isoproterenol was supported by detection of 11 distinct mobility forms of the pentameric protein by use of the Western blotting method, consistent with each phospholamban monomer containing two phosphorylation sites, and with each pentamer containing from 0 to 10 incorporated phosphates. Our results localize the sites of in situ phospholamban phosphorylation to serine 16 and threonine 17 and, furthermore, are consistent with the phosphorylations of these 2 residues being catalyzed by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively.

Adenosine Triphosphatases↗
PubMed1988

Regulation of diacylglycerol kinase reaction in Swiss 3T3 cells. Increased phosphorylation of endogenous diacylglycerol and decreased phosphorylation of didecanoylglycerol in response to platelet-derived growth factor.

We studied the influence of platelet-derived growth factor (PDGF) on diacylglycerol phosphorylation in Swiss 3T3 cells. Rates of incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PtdIns) were determined in prelabeled cells into which sn-1,2-didecanoylglycerol (diC10) had been introduced. PDGF stimulated the formation of [32P]PA and -PtdIns from endogenous substrates but decreased the formation of [32P]PA10 and -PtdIns10. Direct measurements of diacylglycerol phosphorylation in lysates of quiescent and stimulated cells showed that PDGF stimulated the phosphorylation of endogenous diacylglycerol 2-fold in parallel with diacylglycerol accumulation but decreased by 50% the phosphorylation of diC10. Total diacylglycerol kinase activity, measured in a mixed micellar assay, was not changed by PDGF treatment. The maximum activity of diacylglycerol kinase exceeded that needed to phosphorylate all of the endogenous diacylglycerol, suggesting that the PDGF-dependent increase in diacylglycerol mass would account for the increase in PA formation. The increased mass of diacylglycerol also could explain the inhibition of diC10 phosphorylation, via substrate competition. The predominant species of endogenous diacylglycerol was 1-stearoyl-2-arachidonoyl-glycerol (18:0/20:4 diacylglycerol). In mixed micelles, the rate of phosphorylation of 18:0/20:4 diacylglycerol was 8-fold higher than that of diC10, and the 18:0/20:4 species competed with diC10 for phosphorylation. Studies showed that a membrane-bound enzyme accounted for the PDGF effect on PA formation; there was no evidence for translocation of cytosolic enzyme to the membrane. The results support these conclusions: 1) PDGF stimulates the phosphorylation of cellular diacylglycerol by promoting a transient accumulation of this lipid. 2) The stimulated phosphorylation is catalyzed by a diacylglycerol kinase that preferentially phosphorylates 18:0/20:4 diacylglycerol over diC10. 3) The diacylglycerol kinase responsible for the PDGF effect is membrane-bound.

Animals↗