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PubMed1994

Substrate specificity and kinetics of thylakoid phosphoprotein phosphatase reactions.

A synthetic 15-amino-acid phosphopeptide analogue of an N-terminal phosphorylated segment of LHC II was found to inhibit dephosphorylation not only of phospho-LHC II but of all other thylakoid phosphoproteins resolved by phosphorimaging. The results suggest that structural features required for recognition of the phosphoprotein phosphatase are common to different thylakoid phosphoproteins as well as to the phosphopeptide itself: at least one thylakoid phosphoprotein phosphatase exhibits a broad substrate specificity. Dephosphorylation reaction rates of all 13 thylakoid phosphoproteins were determined, and the dephosphorylation half-times were found to range from 7 min to more than 180 min. Most of the phosphoprotein dephosphorylation reactions were partially inhibited by NaF, and were insensitive to antimycin A and okadaic acid. Nevertheless, both antimycin A and NaF stimulated the phosphorylation of LHC II and 9 kDa protein. Possible reasons for differences in sensitivity to these inhibitors are discussed.

Amino Acid Sequence↗

PubMed1996

Cyclic AMP-dependent protein kinase phosphorylates residues in the C-terminal domain of the cardiac L-type calcium channel alpha1 subunit.

The molecular basis of the regulation of cardiac L-type calcium channel activity by cAMP-dependent protein kinase (cA-PK) remains unclear. Direct cA-PK-dependent phosphorylation of the bovine ventricular alpha1 subunit in vitro has been demonstrated in microsomal membranes, detergent extracts and partially purified (+)-[3H]PN 200-110 receptor preparations. Two 32P-labeled phosphopeptides, derived from cyanogen bromide cleavage, of 4.7 and 9.5 kDa were immunoprecipitated specifically by site-directed antibodies against the rabbit cardiac alpha1 subunit amino acid sequences 1602-1616 and 1681-1694, respectively, consistent with phosphorylation at the cA-PK consensus sites at Ser(1627) and Ser(1700). No phosphopeptide products consistent with phosphorylation at three other C-terminal cA-PK consensus phosphorylation sites (Ser(1575), Ser(1848) and Ser(1928)) were identified using similar procedures suggesting that these sites are poor substrates for this kinase. Ser(1627) and Ser(1700) may represent sites of cA-PK phosphorylation involved in the physiological regulation of cardiac L-type calcium channel function.

Amino Acid Sequence↗

PubMed1984

Phorbol ester-induced threonine phosphorylation of the human epidermal growth factor receptor occurs within the EGF binding domain.

Partial proteolysis with trypsin has been used to map the sites of phorbol ester-induced phosphorylation of the epidermal growth factor (EGF) receptor. Both 12-O-tetradecanoylphorbol 13-acetate (TPA) and EGF stimulate phosphorylation of the EGF receptor in intact human carcinoma cells. Under the conditions examined, EGF is more effective than TPA in stimulating phosphorylation of a 45 kDa intracellular receptor domain, while TPA is more effective than EGF in inducing phosphorylation of a 120 kDa transmembrane EGF-binding domain. The phosphorylation of the 120 kDa peptide occurs primarily on threonine residues. Two-dimensional peptide mapping indicates that the two major phosphopeptides found in the 120 kDa receptor fragment correspond to the major new phosphopeptides found in intact EGF receptor following treatment with TPA. Thus, the major sites of TPA-induced threonine phosphorylation reside in the 120 kDa binding domain of the EGF receptor.

Binding Sites↗

PubMed1991

Novel serine phosphorylation occurs in the fibroblast form of pp60c-src from Y79 retinoblastoma cells.

Two-dimensional tryptic peptide analysis showed that pp60c-src from the human retinoblastoma cell line Y79 gave a unique phosphopeptide, which was not found in human fibroblast RT59. There was no significant difference in the extent of phosphorylation of other peptides between the two cell lines. Only phosphoserine was detected in this phosphopeptide. Both the fibroblast form and the neuronal form of pp60c-src from Y79 cells had this unique peptide phosphorylated to the same extent. The phosphorylation site was inferred to be serine 97 by comparing the tryptic map and the arginyl-endopeptidase map. The specific protein kinase activity of pp60c-src from Y79 cells was nearly equal to that of RT59 pp60c-src. This unique serine phosphorylation in the fibroblast form was discussed in relation to the oncogenic change of Y79 cells.

Cell Line↗

PubMed1983

Relationship between H1 histone phosphorylation and genome replication in a mouse-Chinese hamster somatic cell hybrid.

The relationship between stable genome replication and the presence of species-specific H1 and H2b histones was studied in a mouse-Chinese hamster somatic cell hybrid (LMC-1). LMC-1 contained the complete chromosome complements of the mouse (LM/TK-, 46 chromosomes) and Chinese hamster (CHW-1102, 22 chromosomes) parents. No chromosome loss occurred in over a year in continuous culture, and both mouse and Chinese hamster enzymes were expressed. Chromatographic resolution of H1 histone subtypes showed that only mouse H1 genes were expressed. Resolution of chromatographically purified H2b histone variants using Triton X-100-containing gels also demonstrated that LMC-1 cells made mouse, not Chinese hamster, H2b histones. Species-specific tryptic phosphopeptides were resolved from purified H1 histones using high voltage paper electrophoresis. These studies demonstrated that characteristic mouse, but not Chinese hamster, phosphopeptides were formed in H1 histones in actively-growing LMC-1 cells. We conclude that the Chinese hamster genome may replicate in a stable manner in the presence of mouse H1 (and H2b) histones, and that there is no obligatory relationship between species-specific phosphorylation of H1 histones and stable genome replication.

Animals↗

PubMed1983

Metabolite-controlled phosphorylation of hepatic phosphofructokinase proceeds by cAMP-dependent protein kinase.

In hepatocytes 32P-incorporation into rat liver phosphofructokinase is stimulated by glucose as well as by glucagon, the effects of both stimuli being prevented by L-alanine [Eur. J. Biochem. (1982) 122, 175]. The phosphopeptides of the enzyme derived from limited proteolysis by subtilisin and from exhaustive tryptic digestion were analyzed either by one-dimensional mapping on sodium dodecyl sulphate-polyacrylamide slab gels and by fingerprint mapping, respectively. It is shown that in vivo stimulation of 32P-incorporation by glucose or by glucose plus glucagon results in identical phosphopeptide maps, and that these maps were identical with those obtained from phosphofructokinase phosphorylated in vitro with catalytic subunit of cAMP-dependent protein kinase. It is concluded that in the intact liver cell phosphofructokinase is phosphorylated by cAMP-dependent protein kinase but that the state of phosphorylation is modified by metabolite control.

Animals↗

PubMed1983

Presence in many mammalian tissues of an identical major cytosolic substrate (Mr 100 000) for calmodulin-dependent protein kinase.

Incubation of cytosol fractions from a variety of mammalian tissues (heart, liver, lung, adrenal, spleen and skeletal muscle) with Ca2+ (0.5 mM) in the presence of gamma-[32P]ATP resulted in the phosphorylation of a prominent substrate of Mr approximately 100 000 (100 kDa). One-dimensional peptide maps and two-dimensional tryptic fingerprints of the phosphoprotein from these sources were identical. A single major phosphopeptide was generated by trypsin and was determined to contain exclusively phosphothreonine. The 100 kDa substrate could be distinguished from glycogen phosphorylase (Mr approximately 97 000) by a number of criteria including phosphopeptide mapping and by its failure to bind either to glycogen or to a specific antiphosphorylase antibody. The Ca2+-dependent protein kinase responsible for phosphorylation of the 100 kDa protein appeared to be a calmodulin (CaM)-requiring enzyme in that it could be inhibited in cytosol extracts by trifluoperazine (IC50 6-16 microM) and that exogenous CaM was necessary for 100 kDa phosphorylation in CaM-depleted cytosol. These results suggest that a rise in intracellular Ca2+ resulting in an activation of CaM-dependent protein kinase leads to the phosphorylation of a common 100 kDa substrate in many tissues.

Adrenal Glands↗

PubMed1985

Protease-activated kinase II as the mediator of epidermal growth factor-stimulated phosphorylation of ribosomal protein S6.

Epidermal growth factor stimulates phosphorylation of ribosomal protein S6 in serum-starved Swiss 3T3 cells, leading to the formation of highly phosphorylated derivatives containing 4-5 phosphates. Two-dimensional analysis of tryptic phosphopeptides of S6 shows an identical pattern to the ones obtained previously in other cells in response to insulin and the tumor promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate. This suggests a common intracellular mediator of S6 phosphorylation by different growth promoting agents. It is proposed that the potential mediator of this phosphorylation is the Ca2+-independent, cAMP-independent protein kinase, protease activated kinase II, as shown by the extent of phosphorylation and the tryptic phosphopeptide maps of S6 with highly purified enzyme [(1983) J. Biol. Chem. 258, 13998-14002].

Animals↗

PubMed1992

Phosphorylation sequences in h-caldesmon from phorbol ester-stimulated canine aortas.

The high molecular weight form of caldesmon (h-caldesmon) is phosphorylated in vascular smooth muscle. The stoichiometry of caldesmon phosphorylation increases in response to stimulation of the muscle by several contractile agonists; however, the responsible kinase has not been identified. In this study, we have sequenced the phosphopeptides prepared from h-caldesmon phosphorylated in vitro by protein kinase C (PKC) as well as the phosphopeptides prepared from caldesmon phosphorylated in intact canine aortas that were stimulated to contract with PDBu. PKC phosphorylated three sites located in the C terminus: GSS*LKIEE, AEFLNKS*VQK and NLWEKQS*VDK, while h-caldesmon from intact tissue was phosphorylated at two separate sites also in the C terminus: VTS*PTKV and S*PAPK. By comparison to known substrate consensus sequences for various protein kinases these data suggest that h-caldesmon is directly phosphorylated by a proline-directed protein kinase and not by PKC.

Amino Acid Sequence↗

PubMed1993

Interferon-gamma and tumor necrosis factor-alpha enhance p60src expression in human macrophages and myelomonocytic cell lines.

We investigated modulation of p60src expression in human mononuclear phagocytes. By analysis of [35S]methionine-labelled cells we found that synthesis of p60src is higher in human monocytes compared to macrophages derived from in vitro cultivation of monocytes. Western blot analysis showed that expression of p60src in monocyte-derived macrophages can be enhanced if monocytes are differentiated into macrophages in the presence of interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha). Enhanced p60src expression caused by IFN-gamma or TNF-alpha correlated with an enhanced autophosphorylating kinase activity assayed in anti-p60src immune precipitates. In vivo phosphorylation of p60src and analysis of phosphopeptides by tryptic digestion showed that treatment with cytokines did not affect the pattern of phosphorylation of distinct phosphopeptides. The human monocytic cell lines, U937 and HL-60, induced to differentiate along the monocytic pathway by IFN-gamma, or a combination of IFN-gamma and TNF-alpha, expressed higher amounts of the p60src, but not of the p59fyn or p62yes, kinase activity. These findings show that p60src is modulated in the course of differentiation of human monocytes to macrophages, and that macrophage-activating cytokines increase p60src expression in human monocyte-derived macrophages.

Cell Differentiation↗

PubMed1993

Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-Caldesmon.

h-Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologic stimulation. Although many kinases phosphorylate h-caldesmon, in vitro, the responsible kinase in intact tissue is unknown. The sites of phosphorylation in caldesmon from intact canine aortas have recently been identified and are consensus sequences for a proline-directed protein kinase. In this study, we investigated the phosphorylation of h-caldesmon by mitogen-activated protein kinase (MAPK). Purified, recombinant MAPK phosphorylated porcine stomach h-caldesmon to a stoichiometry approaching 2 mol phosphate/mol protein. Phosphorylated h-caldesmon was subjected to proteolysis and the phosphopeptides were purified by high performance liquid chromatography. Two major phosphopeptides were identified and sequenced. These two peptides, VTS*PTKV and S*PAPK, were identical to the sequences of the sites phosphorylated in intact tissue. Antibodies to several enzymes implicated in the cascade of activation of MAPK were used to evaluate vascular smooth muscle by Western blotting. All components were found to be present. These data suggest that MAPK can function as a 'caldesmon kinase' in vascular smooth muscle.

Amino Acid Sequence↗

PubMed1995

S-phase phosphorylation of lamin B2.

Lamin B2 modification in synchronously dividing populations of human diploid fibroblasts was determined by 2-dimensional gel electrophoresis and [32P]orthophosphate labelling. In quiescent (G0) and G1 cultures of HDF, lamin B2 migrated as 2 spots on 2-dimensional gels. In contrast, in S-phase populations of HDF lamin B2 migrated as a single basic species. The level of lamin B2 phosphorylation was determined after immunoisolation from [32P]orthophosphate labelled cells. The results of these experiments indicated a 2-3-fold increase in the steady state level of lamin B2 phosphorylation in S-phase HDF compared with G0 HDF. Consistent with this evidence, tryptic peptide maps revealed the presence of a phosphopeptide in S-phase lamin B2 which was absent from G0 lamin B2. Since all of the phosphate incorporated into S-phase and G0 lamin B2 was recovered in serine residues we conclude that the S-phase specific phosphopeptide did not represent either of the cdc2 sites associated with entry nuclear lamina breakdown.

Blotting, Western↗

PubMed1983

Phosphorylation of proteolytically-nicked rat hepatic acetyl-CoA carboxylase.

A partially-purified preparation of acetyl-CoA carboxylase was not inactivated by ATP and Mg2+ although it was phosphorylated. SDS gel electrophoresis of the phosphorylated enzyme showed phosphopeptides migrating at 140 and 40 K along with the 250 K native subunit. Phosphorylation by the catalytic subunit of cAMP-dependent protein kinase further phosphorylated an additional 120 K phosphopeptide. Neither cAMP-independent phosphorylation nor the cAMP-dependent phosphorylation of the enzyme resulted in a significant decrease in activity.

Acetyl-CoA Carboxylase↗

PubMed1989

Phosphorylation of the human erythrocyte glucose transporter by protein kinase C: localization of the site of in vivo and in vitro phosphorylation.

1. The human erythrocyte glucose transporter was phosphorylated in vitro by protein kinase C. 2. Tryptic cleavage of phosphorylated native transporter produced two major unphosphorylated membrane-embedded fragments weighing 23 and 19 kDa and released numerous water-soluble peptides. 3. Ion-exchange FPLC of the soluble tryptic peptides resolved the mixture into two phosphopeptide peaks. 4. Tryptic digestion of glucose transporter that was phosphorylated in vivo in response to phorbol esters produced soluble phosphopeptides that eluted at identical salt concentrations. 5. Proteolytic digestion and peptide mapping of the transporter revealed that the site(s) of phosphorylation lie within the large cytoplasmic domain that bisects the molecule.

Chromatography, High Pressure Liquid↗

PubMed1986

Phosphorylation of pp60v-src by the TPA receptor kinase (protein kinase C).

Incubation of pp60v-src with the purified 12-O-tetradecanoyl-13-acetate (TPA) receptor kinase (protein kinase C) resulted in an increase in the phosphorylation of pp60v-src. Two-dimensional tryptic phosphopeptide mapping showed that three major phosphoserine containing peptides were labeled which were localized within the amino terminal 18,000 Da of the src protein. Based on comparative tryptic mapping, one of these major phosphopeptides was identical to the peptide labeled on pp60v-src immunoprecipitated from cells labeled with [32P]orthophosphate and treated with TPA. These data suggest that a direct interaction takes place between protein kinase C and pp60v-src.

Animals↗

PubMed1992

Characterization of the phosphorylated state of protein 4.2 from a patient partially deficient in protein 4.2.

These studies compare the protein 4.2 found in a patient with osmotically fragile, spherocytic erythrocytes to the normal protein 4.2. The patient protein 4.2 is present in the erythrocyte ghost membranes as a doublet of 74 and 72 KDa at a concentration less than 1% of normal. The patient protein 4.2 becomes highly phosphorylated in the presence of Zn++ and is phosphorylated, relative to the amount of protein present, to a greater extent than the normal 72 KDa protein 4.2. These studies indicate that both the patient and the normal protein 4.2 usually exists in a highly phosphorylated state. The phosphorylation sites on the patient protein 4.2 appear to be more readily cycled than on the normal protein 4.2. Staphylococcus aureus V8 protease generates similar phosphopeptides in both the normal and patient protein 4.2 except for an extra 11 KDa phosphopeptide generated from the 74 KDa form of the protein.

Blood Proteins↗

PubMed1982

Dephosphorylation of glycogen synthase from human polymorphonuclear leukocytes.

Leukocyte glycogen synthase (UDPglucose:glycogen 4-alpha-d-glucosyltransferase, EC 2.4.1.11) was phosphorylated to about one P1/synthase subunit by either the cAMP-dependent protein kinase or the cAMP-dependent synthase kinase. The relationship between dephosphorylation and the increase in the ratio of independence was investigated by analysis of the release of 32P-labelled phosphopeptides from the trypsin-sensitive and the trypsin-insensitive regions. The trypsin-insensitive region was predominantly dephosphorylated and a close correlation between dephosphorylation of a phosphopeptide in the trypsin-insensitive region and activation of glycogen synthase is reported for the enzyme phosphorylated in both ways.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1986

Regulation of the phosphorylation state and function of the epidermal growth factor receptor by vitamin K-3.

Vitamin K-3 or 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced the binding of epidermal growth factor (EGF) to its receptor by more than 90% in human foreskin fibroblasts. After the equilibration of fibroblasts with [32P]orthophosphate, vitamin K-3 or TPA markedly increased the amount of 32P found in the receptor; the increase was principally due to serine and threonine phosphorylation. By the use of two-dimensional tryptic phosphopeptide mapping, using a synthetic phosphopeptide as a standard, threonine-654 was identified as one of the residues whose phosphorylation state was elevated by vitamin K-3 or TPA. Because of the large amounts of EGF receptor present on A431 human carcinoma cells, these cells were used to study further the relationship between the phosphorylation state of threonine-654, the tyrosine phosphorylation state of the receptor, and the receptor's protein tyrosine kinase activity toward exogenous substrates. Vitamin K-3 and TPA both increased the amount of phosphate on threonine-654 in A431 cells. However, whereas receptor from TPA-treated cells lacked phosphotyrosine, vitamin K-3-treated cells contained receptor with markedly elevated levels of phosphotyrosine. The addition of vitamin K-3, TPA or EGF to intact A431 cells followed by homogenization of the cells and the assay of EGF receptor protein tyrosine kinase activity by the use of a synthetic peptide substrate resulted in marked decreases in apparent receptor kinase activity. Therefore, assuming that the activity measured in the peptide assay reflects the protein tyrosine kinase activity of the receptor in the intact cell, the activity of the EGF receptor kinase cannot be deduced from the amount of phosphotyrosine associated with the receptor.

Calcium↗