SEARCH · PubMed Health

Results for “PHOSPHOPEPTIDES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 739 records · Page 41Linked to original sources

PubMed2005

Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides.

Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.

Affinity Labels↗

PubMed2004

An efficient protocol for the identification of protein phosphorylation in a seedless plant, sensitive enough to detect members of signalling cascades.

We describe a reproducible protocol to explore for the first time the phosphoproteome of a seedless plant, the moss Physcomitrella patens. Following tryptic digestion of a total protein extract, phosphorylated peptides were isolated using the combination of C18 reverse-phase chromatography (RP-C18), immobilized Fe(3+) metal affinity chromatography (IMAC), capillary zone electrophoresis (CZE), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. The total protein extracts were first prepared as usually made for plant two-dimensional gel electrophoresis, the tryptic digest was desalted and concentrated by reverse phase chromatography, and from this mixture the phosphorylated peptides were captured by IMAC. Subsequently, the complex phosphopeptide mixture was separated into ten fractions by RP-C18-HPLC and each analyzed by CZE. This permitted the detection of 253 distinct phosphopeptides. These were identified by nano-LC-MS/MS and MALDI-TOF-MS analysis in conjunction with alkaline phosphatase treatment to remove covalently bound phosphate to specifically identify the phosphopeptides. Among others, several kinases and a transcription factor were identified. This protocol will be taken as a basis to unravel early events in plant signal transduction known to occur via rapid phosphorylation/dephosphorylation of proteins.

Bryopsida↗

PubMed1994

Chromatographic and mass spectrometric methods for the identification of phosphorylation sites in phosphoproteins.

The phosphorylation sites in a model phosphoprotein, alpha s1-casein from bovine milk, have been identified by tryptic peptide mapping (Gibson and Cohen, Methods Enzymol. vol. 193, p. 480 (1990)) employing reversed-phase high performance liquid chromatography (RPHPLC)/electrospray ionization mass spectrometry (ES-MS); by infusion tandem mass spectrometry (MS/MS) and LC/MS/MS in neutral loss mode of tryptic digests of alpha s1-casein, in which the characteristic neutral loss of phosphoric acid by phosphopeptides under collision-induced dissociation (CID) conditions is exploited to highlight phosphopeptides in a tryptic digest (Covey et al., in Methods in Protein Sequence Analysis, Jörnvall et al. (Eds), Birkhäuser Verlag, Basel 1991), and by a novel method, termed LC/CID-MS, in which phosphopeptides are located in mixtures of peptides by the generation and detection of phosphate-specific fragment ions during LC/ES-MS (Huddleston et al., J. Am. Soc. Mass Spectrom. vol. 4, p. 710 (1993)). An appraisal of the efficiency, sensitivity and practicality of each of these methods in the identification of phosphorylation sites in post-translationally modified proteins is given.

Alkaline Phosphatase↗

PubMed1996

Molecular cloning and characterization of a novel casein kinase II substrate, HASPP28, from rat brain.

HASPP28 (heat- and acid-stable phosphoprotein of 28 kDa) has been purified to near homogeneity from the acid-stable protein fraction of rat brain extract. Based on the N-terminal 40 amino acid sequence, a pair of highly degenerate primers was used to generate a 107-bp probe from rat brain RNA by RT-PCR. From the rat brain lambda gt11 library, this probe identified two positive clones that together provided a cDNA of 837 bp with an open reading frame of 546 bp. This cDNA was extended by 3'RACE to 1.2 kb that included a polyadenylation signal and a poly(A) tail. The 180-amino-acid sequence derived from the open reading frame, which did not correspond to any known protein, was predicted to have phosphorylation sites for protein kinase C, casein kinase II (CKII), and protein kinase A. Indeed, both the purified rat brain HASPP28 and the recombinant HASPP28 (rHASPP28) can be phosphorylated by these kinases. Northern blot analysis indicated that HASPP28 was present in all rat tissues tested, including those from the brain, lung, spleen, kidney, liver, heart, and muscle, in decreasing order of abundance. Phosphopeptide analysis of rHASPP28 phosphorylated in vitro by various kinases showed different tryptic patterns on two-dimensional mapping and isoelectric focusing gels. From [32P]PO4-labeled N1E115 neuroblastoma cells, HASPP28 can be immunoprecipitated with a polyclonal antiserum raised against rHASPP28. The immunoprecipitated protein showed a phosphopeptide pattern similar to that of rHASPP28 phosphorylated by CK II in vitro. Furthermore, the immunoprecipitates from cells treated with phorbol 12-myristate 13-acetate or 8-bromo-cAMP did not show any increased phosphorylation over those of untreated ones, and the phosphopeptide patterns of the immunoprecipitates again were similar to that of CK II phosphorylated protein. These results suggest that HASPP28 is a novel phosphoprotein that can be phosphorylated by several kinases in vitro. In intact cells, CK II seems to be solely responsible for the phosphorylation of HASPP28.

Amino Acid Sequence↗

PubMed1994

Dithiothreitol stimulates insulin receptor autophosphorylation at the juxtamembrane domain.

Autophosphorylation of insulin receptors from human placental membranes prepared with iodoacetamide was more than double that of control receptors prepared without iodoacetamide. Dithiothreitol (DTT) treatment of control receptors prepared without iodoacetamide resulted in increased autophosphorylation. However, DTT was without effect on insulin receptors prepared with iodoacetamide. Phosphopeptide analysis showed that while 32P-labeling of all of the phosphopeptides was increased in insulin receptors from membranes prepared with iodoacetamide, two additional phosphopeptides were detected and identified as deriving from the juxtamembrane domain, containing tyrosine residue 960. Similar results were produced by DTT treatment of control insulin receptors. These observations suggest that a thiol(s) may be involved in insulin receptor autophosphorylation in the juxtamembrane domain.

Dithiothreitol↗

PubMed1997

Phosphorylation of connexin43 and the regulation of neonatal rat cardiac myocyte gap junctions.

The functional state of gap junctions and the state of phosphorylation of connexin43 (Cx43), the major gap junction protein in rat heart, were evaluated in primary cultures of neonatal rat cardiocytes. Functional coupling was greatly reduced after treatment with staurosporine (ST), a protein kinase inhibitor. The ST-induced reduction in cell coupling was reversed by activation of protein kinase C (PKC) with 12-O-tetradecanoylphorbol 13-acetate (TPA). The cellular distribution of Cx43, as detected by immunofluorescence, was not grossly affected by either ST alone or ST plus TPA. Although immunoblot analysis did not detect significant changes in the relative amounts of the unphosphorylated and individual phosphorylated forms of Cx43 after each treatment, the level of 32P-incorporation into Cx43 of radiolabeled cells was significantly affected. Consistent with their known properties, treatment with ST reduced, and combined treatment with TPA and ST increased, the level of 32P-incorporation into Cx43. Two-dimensional tryptic phosphopeptide maps of 32P-labeled Cx43 indicated that a distinct subset of the phosphopeptides that are present under basal conditions were affected by ST or ST/TPA treatments, with TPA-induced phosphorylation occurring at the ST-sensitive sites. However, the ST/TPA-sensitive tryptic phosphopeptides did not comigrate with others that were derived from in vitro phosphorylation by PKC of a recombinant C-terminal Cx43 peptide (Cx43[243-382]). Although a PKC-dependent mechanism appears to be involved in the regulation of functional coupling between neonatal rat cardiocytes, PKC itself may not be the final mediator of Cx43 phosphorylation.

Animals↗

PubMed1994

Differential sensitivity to inhibitors discriminates between two types of kinases responsible for in vivo phosphorylation of different sites in the carboxy-terminal tail of chicken neurofilament-M.

In order to characterize the phosphorylation of neurofilaments (NF) in intact neurons, we examined the ability of several protein kinase inhibitors to interfere with the incorporation 32P into individual NF polypeptides of sensory neurons in culture. We also examined their effect on the post-translational mobility shift on SDS-PAGE that accompanies phosphorylation of newly synthesized NF-M. Several agents known to inhibit cyclic nucleotide-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases (H7, HA1004, trifluoperizine, sphingosine) had no effect on the phosphorylation of any NF polypeptide, in either assay. In contrast, two broadly active protein kinase inhibitors, staurosporine and K252a, inhibited the incorporation of 32P into NF-M by 60-70% and also blocked the post-translational mobility shift. They had no effect on NF-L. The action of staurosporine and K252a was identical to that of 25 mM LiCl. Proteolytic cleavage and phosphopeptide mapping of 32P-labeled NF-M from control and treated cultures revealed that the phosphorylation of only one subset of phosphopeptides was affected by staurosporine, K252a, and LiCl. These were contained within a single chymotryptic fragment of the NF-M tail segment, probably containing most of the 17 repeats of a KXXS/TP motif. The phosphorylation of another subset of phosphopeptides was insensitive to these inhibitors. They were contained within a different chymotryptic fragment of the tail segment which contains a KSD and four KSP potential phosphorylation sites. This differential sensitivity to protein kinase inhibitors distinguishes two different types of effector-independent kinases that phosphorylate, in vivo, different sites within the NF-M tail.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Intracellular phosphorylation of the Sendai virus P protein.

Phosphorylation status of the Sendai virus P protein was examined during virus infection and compared with cell-free phosphorylation. P protein from Sendai virus-infected (VI) and P/C gene-transfected (PT) mammalian cells and from purified virions (PV) was phosphorylated at only serine residues. In contrast, cell-free phosphorylation of the P protein with virion-associated protein kinase (VAPK) occurred at both threonine and serine. Tryptic phosphopeptide maps of the P protein from VI, PT, and PV showed that the phosphorylation was primarily localized on one peptide (TP1), while VAPK phosphorylated the P protein on several peptides. There was no change in the steady-state phosphopeptide map of the P protein during virus replication, indicating that the TP1 is constitutively phosphorylated. Inhibition of cellular phosphatases (PP1 and PP2A) by okadaic acid (OA) in virus-infected cells caused a sixfold increase in the P protein phosphorylation, solely at serine residues. The phosphopeptide map of the OA-P protein revealed that phosphorylation occurred on several peptides, but the OA-P map was significantly different from the VAPK-P map. However, additional phosphorylation of the P protein did not block its association with nucleocapsids. These results suggest that the Sendai virus P protein is constitutively phosphorylated primarily at one locus but has the potential for phosphorylation at additional sites. Further, our results do not show any correlations between the intracellular and cell-free phosphorylation of the P protein and, therefore, question the validity of cell-free phosphorylations.

Animals↗

PubMed1991

Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells.

The regulation of the plasma membrane Ca2+ pump by hormones via phosphorylation in intact cells has not been clearly established. We now present evidence that the Ca2+ pump is phosphorylated on both serine and threonine residues in unstimulated and stimulated cultured rat aortic endothelial cells. Among the stimuli tested, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was most potent and increased the level of phosphorylation threefold, while the cAMP-dependent protein kinase activator 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated the phosphorylation 1.6-fold. Two-dimensional tryptic phosphopeptide maps of the Ca2+ pump from unstimulated and CPT-cAMP-stimulated cells have identical patterns (five phosphopeptides) while PMA-stimulated cells have three additional phosphopeptides. Isoproterenol-, ATP-, angiotensin II-, and bradykinin-stimulated cells also have increased levels of Ca2+ pump phosphorylation. Stimuli-induced phosphorylation of the Ca2+ pump was rapid (5-10 min) and was concomitant with stimulated calcium efflux from the same cells. This is the first direct evidence that the plasma membrane Ca2+ pump in intact cells is regulated by various hormones or agonists via cAMP-dependent protein kinase or protein kinase C phosphorylation.

Animals↗

PubMed1978

Comparison of two forms of pig heart phosphoprotein phosphatase.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) was partially purified from pig heart using as substrate H2B histone which had been phosphorylated at Ser-32 and Ser-36 by adenosine 3',5'-monophosphate-dependent protein kinase (EC 2.7.1.37). The enzyme had a molecular weight of approx. 250 000 and was converted to a smaller form with a molecular weight of approx. 30 000 upon treatment with ethanol. Phosphorylase alpha (EC 2.4.1.1) and phosphorylated H1 histone also served as substrates for both forms of the enzyme. The conversion of the large form of the enzyme to the small form decreased the phosphohistone phosphatase activity to 25-50% with a concomitant 7-fold increase in the phosphorylase alpha phosphatase activity. Ser-36 phosphate was removed 6- and 15-fold more rapidly than was Ser-32 phosphate by the large and small forms of the enzyme, respectively. Among Ser-36-containing tryptic phosphopeptides derived from phosphorylated H2B histone, Lys-Glu-Ser(P)-Tyr-Ser-Val-Tyr was the shortest phosphopeptide which was dephosphorylated at a significant reaction rate with the phosphoprotein phosphatase. The Km values for phosphorylated H2B histone and the tryptic phosphopeptide were 23.7 micron and 187.1 micron, respectively, with the large form, and 81.4 micron and 90.0 micron, respectively, with the small form of the enzyme.

Animals↗

PubMed1986

Evidence that (a) serine specific protein kinase(s) different from protein kinase C is responsible for the insulin-stimulated actin phosphorylation by placental membrane.

Partially purified phospholipid- and Ca2+-dependent protein kinase C from human placenta catalyzes the Mg-ATP-dependent phosphorylation of serine residues of purified rabbit muscle actin. Two tryptic [32P]-phosphopeptides were found on HPLC separation. Confirming the previous report by Machicao and Wieland [(1985) Curr. Top. Cell. Regul. 27, 95-105], actin is phosphorylated at serine residues by human placental membranes, and this is stimulated by insulin. In the absence of insulin trypsin treatment yielded eight [32P]phosphopeptides, two of which coincided with the ones due to protein kinase C. Insulin led to the appearance of three new [32P]phosphopeptides. These results suggest that insulin stimulates (a) serine protein kinase(s) which, like protein kinase C, is present in placental membranes.

Actins↗

PubMed1989

Identification of a phosphorylation site of the rat insulin receptor catalyzed by protein kinase C in an intact cell.

In two-dimensional tryptic phosphopeptide mapping, the beta-subunit of the insulin receptor phosphorylated by 12-O-tetradecanoylphorbol-13-acetate in rat hepatoma cells (H-35) was separated into one phosphothreonine-containing peptide and several phosphoserine-containing peptides. The synthetic peptide coding residues 1327-1343 in the C-terminal region of the rat insulin receptor was phosphorylated at the threonine residue by protein kinase C in a phosphatidylserine and oleoylacetylglycerol dependent manner. Tryptic digest of this phosphopeptide migrated to the same position as the phosphothreonine containing peptide obtained from the beta-subunit in two-dimensional phosphopeptide mapping. These data suggested that Thr 1336 of the insulin receptor is the site of phosphorylation by protein kinase C in intact cells.

Animals↗

PubMed1991

Dephosphin, a 96,000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes.

A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.

Animals↗

PubMed1993

Protein phosphatase assay using a modification of the P81 paper protein kinase assay procedure.

Synthetic peptides have been used to define specificity determinants and to distinguish reactivities of numerous protein kinases and phosphoprotein phosphatases. Direct analysis of peptide phosphorylation is most often determined using P81 phosphocellulose paper to separate modified peptide and unreacted [gamma-32P]ATP; however phosphopeptide dephosphorylation is usually determined by extraction and quantitation of phosphomolybdate complexes or ion exchange chromatography. We describe here the adaptation of the rapid, direct P81 paper protein kinase assay for the determination of phosphopeptide dephosphorylation. The S6-21 peptide (AKRRRLSSLRASTSKSESSQK), which is derived from the multiphosphorylated carboxyl terminal domain of the S6 ribosomal protein, was phosphorylated by a human placenta S6 kinase and dephosphorylation by purified phosphoprotein phosphatase type 1 in the presence of a variety of buffers, and inhibitors/activators was determined using the new assay. Results comparable to those obtained with the ion-exchange chromatography were obtained, and the assay was significantly less expensive, more rapid, and more accurate than methods previously used to quantitate phosphopeptide dephosphorylation.

Amino Acid Sequence↗

PubMed1988

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit.

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.

Amino Acid Sequence↗

PubMed1989

Casein kinase-2 phosphorylates serine-2 in the beta-subunit of initiation factor-2.

We have previously presented evidence which suggests that casein kinase-2 phosphorylates a serine residue near the N-terminus of the beta-subunit of the initiation factor eIF-2 (Clark, S.J. et al. Biochim. Biophys. Acta 968, 211-219). We now report further data which confirm that it is serine-2 which is phosphorylated by casein kinase-2. This data includes (1) the electrophoretic mobilities of the phosphopeptides produced by different cleavage techniques, (2) the amino acid composition of the principal phosphopeptide generated by treatment with cyanogen bromide and (3) the resistance of this phosphopeptide to Edman degradation.

Amino Acids↗

PubMed1993

Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase.

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.

Amino Acid Sequence↗

PubMed1994

Arginine vasopressin (AVP) causes the reversible phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in the ovine anterior pituitary: evidence that MARCKS phosphorylation is associated with adrenocorticotropin (ACTH) secretion.

We have recently shown that AVP causes a protein kinase C (PKC)-dependent increase in ACTH release and biosynthesis in ovine anterior pituitary cells. In these cells, AVP also causes the translocation of PKC from the cytosol to the cell membrane which is maximal at 5 min, but the intracellular events distal to protein kinase C activation that underlie ACTH secretion have not been well characterized to date. Since the MARCKS protein has been implicated in neurosecretion and is phosphorylated by PKC in synaptosomes, studies were carried out to determine whether AVP might cause MARCKS phosphorylation in the ovine anterior pituitary, and to determine whether this phenomenon might be temporally correlated with PKC translocation and the release of ACTH. When cytosolic fractions of rat brain, ovine anterior pituitary, and cultured ovine anterior pituitary cells were incubated with purified PKC, several proteins were phosphorylated including those in the region of 83-85 kDa. After precipitation of the proteins with 40% acetic acid, the 83-85 kDa phosphoproteins were selectively recovered in the acid soluble phase. Phosphopeptide maps of either the 83 or 85 kDa proteins were generated with Staphylococcus aureus V8 protease and revealed 13 and 9 kDa phosphopeptides, which are characteristic of the authentic MARCKS protein. An identical phosphopeptide map was also obtained when the MARCKS protein was selectively extracted from intact 32P-labeled anterior pituitary cells. MARCKS phosphorylation was markedly increased when ovine anterior pituitary cells were exposed to 1 microM phorbol 12-myristate 13-acetate (PMA). When the cells were exposed to 1 microM AVP, MARCKS phosphorylation increased at 15 s and reached the maximal plateau value at 30 s. MARCKS phosphorylation then started to diminish at 2 min, and baseline levels were attained by 10 min. In the same cells, AVP stimulated ACTH release in a biphasic manner-during the first 30 s, there resulted a rapid burst of ACTH secretion that was followed by a slower, but sustained rate of secretion. We conclude that: (1) AVP causes a rapid, and reversible, phosphorylation of the MARCKS protein in the ovine anterior pituitary; (2) since the AVP-induced increase in MARCKS phosphorylation occurs much earlier in these cells than does PKC trans-location, MARCKS phosphorylation may provide a more sensitive index of the onset of PKC activation than the translocation assay; (3) the close temporal association between MARCKS phosphorylation and the rapid early release of ACTH suggests that MARCKS phosphorylation may be involved in the initial intracellular events that underly exocytosis of the hormone.

Adrenocorticotropic Hormone↗