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PubMed2005

Differential multisite phosphorylation of the trehalose-6-phosphate synthase gene family in Arabidopsis thaliana: a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis.

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.

Amino Acid Sequence↗

PubMed2006

Fish-bone peptide increases calcium solubility and bioavailability in ovariectomised rats.

Fish-bone peptides (FBP) with a high affinity to Ca were isolated using hydroxyapatite affinity chromatography, and FBP II with a high ratio of phosphopeptide was fractionated in the range of molecular weight 5.0-1.0 kDa by ultramembrane filtration. In vitro study elucidated that FBP II could inhibit the formation of insoluble Ca salts in neutral pH. In vivo effects of FBP II on Ca bioavailability were further examined in the ovariectomised rat. During the experimental period, Ca retention was increased and loss of bone mineral was decreased by FBP II supplementation in ovariectomised rats. After the low-Ca diet, the FBP II diet, including both normal level of Ca and vitamin D, significantly decreased Ca loss in faeces and increased Ca retention compared with the control diet. The levels of femoral total Ca, bone mineral density, and strength were also significantly increased by the FBP II diet to levels similar to those of the casein phosphopeptide diet group (no difference; P>0.05). In the present study, the results proved the beneficial effects of fish-meal in preventing Ca deficiency due to increased Ca bioavailability by FBP intake.

Absorption↗

PubMed1997

cAMP-dependent phosphorylation activates phosphofructokinase from mantle tissue of the mollusc Mytilus galloprovincialis. Identification of the phosphorylated site.

Phosphofructokinase from mantle tissue of the sea mussel Mytilus galloprovincialis was phosphorylated in vitro by a protein kinase isolated from the same tissue, homologous to mammalian cAMP-dependent protein kinase; the maximal level of phosphorylation achieved was around 1 mol of Pi/mol of phosphofructokinase subunit. The covalent incorporation of phosphate leads to a notable increase in the enzyme activity assayed at near-physiological concentrations of substrates and allosteric modulators and neutral pH. Tryptic digestion of labeled phosphofructokinase released a phosphopeptide whose sequence was Lys-Asp-Ser(P)-Ile-Trp-Ile-Gln-Thr-Gly-Arg. This sequence showed high homology with the phosphopeptides from other invertebrates whose phosphofructokinase is also activated by cAMP-dependent phosphorylation.

Amino Acid Sequence↗

PubMed1973

Further characterization of the F1-histone phosphokinase of metaphase-arrested animal cells.

Exponentially growing Chinese hamster cells are found to contain two major phosphokinase activities with specificity for the phosphorylation of F1 (lysine-rich) histone. These two activities, designated KI and KII, were extracted with 0.35 M NaCl and fractionated in 0.2 M NaCl by Sephadex G-200 gel filtration. KI, which is similar to the ubiquitous cyclic 3',5'-adenosine monophosphate (cAMP)-dependent phosphokinase, differs from KII by several criteria. KII is mol wt 90,000, cAMP independent, rapidly turned over in vivo, low K(m) for ATP, and phosphorylates F1 histone at several unique sites. Comparative examination of metaphase-arrested (M) and counterpart interphase (I) cells for these two activities reveals that KII is responsible for the overall high activity in M-arrested cells. Pulse labeling of cells with (32)P during traverse of the G(2)-M phase of the cell cycle reveals an in vivo tryptic-phosphopeptide pattern in whole unfractionated F1 which is unique to M cells. Seven major phosphopeptides derived by in vitro phosphorylation of F1 with the KII enzyme correspond to these M cell-specific phosphorylation sites observed in vivo. It is suggested that KII activity predominates during the G(2)-M transition and that F1 is its natural in vivo substrate.

Animals↗

PubMed2000

Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI.

The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).

Amino Acid Sequence↗

PubMed2006

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism.

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.

Amino Acid Motifs↗

PubMed2000

Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors.

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.

14-3-3 Proteins↗

PubMed2000

The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.

Amino Acid Sequence↗

PubMed2005

PrpZ, a Salmonella enterica serovar Typhi serine/threonine protein phosphatase 2C with dual substrate specificity.

Genes encoding eukaryotic-type protein kinases and phosphatases are present in many bacterial genomes. An ORF encoding a polypeptide with homology to protein phosphatases 2C (PP2Cs) was identified in the genomes of Salmonella enterica serovar Typhi strains CT18 and Ty2. This protein, termed PrpZ, is the first PP2C to be identified in enterobacteria. Analysis of the amino acid sequence revealed two distinct domains: the N-terminal segment containing motifs of the catalytic domain of PP2Cs and the C-terminal segment with unknown function. PrpZ was expressed in Escherichia coli as a histidine-tagged fusion protein (PrpZ(His)) and the purified protein was analysed for its ability to dephosphorylate various substrates. Using p-nitrophenyl phosphate as a substrate, optimal PrpZ(His) activity was observed at pH 9.5, with a strong preference for Mn(2+) over Mg(2+). Activity of PrpZ(His) was inhibited by EDTA, sodium fluoride, sodium phosphate and sodium pyrophosphate but unaffected by okadaic acid, indicating that PrpZ is a PP2C. Using synthetic phosphopeptides as substrates, PrpZ(His) could hydrolyse phosphorylated serine, threonine or tyrosine residues, with the highest catalytic efficiency (k(cat)/K(m)) for the threonine phosphopeptide. With phosphorylated myelin basic protein (MBP) as the substrate, Mn(2+) was only twofold more efficient than Mg(2+) in stimulating PrpZ(His) activity at pH 8.0. The ability of PrpZ(His) to remove the phosphoryl group from phosphotyrosine residues was confirmed by measuring the release of inorganic phosphate from phospho-Tyr MBP. Together, these data indicate that PrpZ has all the features of a PP2C with dual substrate specificity in vitro.

Base Sequence↗

PubMed2002

In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase.

Sucrose synthase (SS) is a known phosphoserine (SerP)-containing enzyme in a variety of plant "sink" organs, including legume root nodules, where it is phosphorylated primarily at Ser-11. Using immunofluorescence confocal microscopy, we documented that part of the total SS (nodulin-100) pool in mature soybean (Glycine max) nodules is apparently associated with the plasma membrane in situ, and we report that this association is very "tight," as evidenced by a variety of chemical and enzymatic pretreatments of the isolated microsomal fraction. To investigate the in situ and in planta phosphorylation state of the membrane (m) and soluble (s) forms of nodule SS, three complementary approaches were used. First, excised nodules were radiolabeled in situ with [(32)P]Pi for subsequent analysis of phosphorylated m- and s-SS; second, immunopurified s- and m-SS were used as substrate in "on-bead" assays of phosphorylation by nodule Ca(2+)-dependent protein kinase; and third, SS-Ser-11(P) phosphopeptide-specific antibodies were developed and used. The collective results provide convincing evidence that microsomal nodulin-100 is phosphorylated in mature nodules, and that it is hypophosphorylated relative to s-SS (on an equivalent SS protein basis) in attached, unstressed nodules. Moreover, the immunological data and related phosphopeptide mapping analyses indicate that a homologous N-terminal seryl-phosphorylation domain and site reside in microsomal nodulin-100. We also observed that mild, short-term inorganic nitrogen and salt stresses have a significant negative impact on the content and N-terminal phosphorylation state of nodule m- and s-SS, with the former being the more sensitive of the two SS forms.

Ammonium Chloride↗

PubMed2000

Comparison of binding energies of SrcSH2-phosphotyrosyl peptides with structure-based prediction using surface area based empirical parameterization.

The prediction of binding energies from the three-dimensional (3D) structure of a protein-ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent-accessible surface area-based calculations for the prediction of the binding of Src-SH2 domain with a series of tyrosyl phosphopeptides based on the high-affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+ 3 position has been changed. Two other peptides based on the C-terminal regulatory site of the Src protein and the platelet-derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area-based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (deltaCp, deltaS, deltaH, and deltaG) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent-accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters: however, the tolerance of this approach is not sufficient to make it ubiquitously applicable.

Amino Acid Sequence↗

PubMed2000

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1996

Solid-phase synthesis of H- and methylphosphonopeptides.

We introduce solid-phase syntheses of H- and methylphosphonopeptides, giving access for the first time to a new class of mimics for o-phosphoamino acids. The model peptides H-GlyGlyXaaAla-OH (Xaa = Ser, Thr) were synthesized on a solid-phase using Fmoc/tBu strategy and HBTU/HOBt activation by incorporation of hydroxyl-protected serine and threonine. As selectively cleavable hydroxyl-protecting groups we used triphenylmethyl and tert-butyldimethylsilyl for both amino acids, as described in the literature. All peptides were phosphitilated with O, O-di-tert-butyl-N,N-diethylphosphoramidite and yielded H-phosphonopeptides after trifluoroacetic acid cleavage. Alternatively we phosphonylated the peptides with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite, which was synthesized by a two-step one-pot procedure starting from commercially available chemicals. All H- and methylphosphonopeptides were obtained in high purities and yields, as shown by reversed-phase high-performance liquid chromatography and anion-exchange chromatography. The phosphonopeptides were characterized by 1H and 31P NMR. We confirmed their molecular masses by electrospray mass spectrometry and analyzed their fragmentation schemes, which seemed to be characteristic for each class of analogues. The H-phosphonopeptides lost phosphonic acid (H3PO3, 82 mass units) and the methylphosphonopeptides lost methylphosphonic acid (MeH2PO3, 96 mass units). Both H- and methylphosphonopeptides represent a new and simply accessible class of mimics for phosphopeptides. Compared with the corresponding phosphopeptides all phosphonopeptides were synthesized in higher yields and purities (> 80%).

Magnetic Resonance Spectroscopy↗

PubMed1987

Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the alpha subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor.

Eukaryotic protein synthesis initiation factor 2 (eIF-2) can be phosphorylated on its alpha subunit by two well-characterised protein kinases, termed the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). Phosphorylation of eIF-2 by these kinases is thought to be important in the regulation of peptide-chain initiation. We report the location of the serine residue in the alpha subunit, which is phosphorylated by both these enzymes. Limited tryptic digestion and subsequent cyanogen bromide treatment of rat liver eIF-2 phosphorylated by HCR yielded one major phosphopeptide. This peptide had the sequence Ile-Leu-Leu-Ser-Glu-Leu-Ser(P)-Arg-Arg. The same major phosphopeptide was obtained from rabbit reticulocyte eIF-2 phosphorylated by HCR or dsI as judged by its behaviour on two-dimensional mapping and reverse-phase chromatography. In all cases the phosphorylated residue was found to be serine-7, and not serine-4, of the above sequence as determined from sequence analysis and by subdigestion of the peptide with Staphylococcus aureus V8 proteinase.

Amino Acid Sequence↗

PubMed1988

Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase.

We have examined the sites phosphorylated on acetyl-CoA carboxylase by three protein kinases which have been shown to inactivate the enzyme, i.e. cyclic-AMP-dependent protein kinase, acetyl-CoA carboxylase kinase-2 (ACK2, purified from rat mammary gland) and the AMP-activated protein kinase (formerly called acetyl-CoA carboxylase kinase-3, purified from rat liver). Each protein kinase phosphorylates two out of three sites (termed 1-3) which have been established by amino acid sequencing. The two sites phosphorylated by each kinase can be recovered on separate peptides, TC1 and TC2, derived by combined digestion of the native enzyme by trypsin and chymotrypsin: TC1 = Ser-2Ser(P)-Met-3Ser(P)-Gly-Leu; TC2 = Arg-Met-1Ser(P)-Phe- Cyclic-AMP-dependent protein kinase phosphorylates sites 1 and 2 exclusively, whereas the AMP-activated protein kinase phosphorylates sites 1 and 3, plus at least one other minor site. ACK2 phosphorylates site 1 and, more slowly, an unidentified site(s) within TC1. We have also established the structures of the single major phosphopeptides (T1 and C1 respectively) which are recovered by HPLC after acetyl-CoA carboxylase phosphorylated by cyclic-AMP-dependent protein kinase is digested with trypsin or chymotrypsin alone. T1 is related to TC1, and has the structure: Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys. C1 is identical with TC2. We have carried out studies on the correlation of the activity of acetyl-CoA carboxylase with the occupancy of sites 1, 2 and 3 during phosphorylation by each of the three protein kinases. The results suggest that phosphorylation of site 3 is primarily responsible for the large decrease in Vmax produced by the AMP-activated protein kinase, while phosphorylation of site 1 may be primarily responsible for the increase in A0.5 for citrate and more modest depression of Vmax produced by cyclic-AMP-dependent protein kinase and ACK2. Our results emphasize that amino acid sequence information is essential in the unequivocal interpretation of data from phosphopeptide mapping experiments and allow a more complete interpretation of previous data on phosphorylation of acetyl-CoA carboxylase in intact cells. They also open the way to experiments which could establish the physiological roles of these protein kinases in the control of fatty acid synthesis.

Acetyl-CoA Carboxylase↗

PubMed1988

Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase.

We have examined the sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P-labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the peptide containing the sites phosphorylated in vitro by casein kinase-2. The other may correspond to the 'I' site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme-bound phosphate (equivalent to less than 0.1 mol phosphate/mol 240-kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic-AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl-CoA carboxylase derived from control and insulin-treated adipocytes, and also after phosphorylation in vitro with casein kinase-2 and the calmodulin-dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe-Ile-Ile-Gly-Ser4-Val-Ser5-Gln-Asp-Asn-Ser6-Glu-Asp -Glu-Ile-Ser-Asn-Leu-. Site 5 was phosphorylated by the calmodulin-dependent protein kinase and site 6 by casein kinase-2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P-labelling of T4a. We were unable to isolate sufficient quantities of the other insulin-sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase-2, or a protein kinase which has the same specificity as casein kinase-2. The function of this modification is not clear, since phosphorylation by casein kinase-2 has no direct effect on acetyl-CoA carboxylase activity.

Acetyl-CoA Carboxylase↗

PubMed1985

Phosphorylation of microtubule-associated protein 2 at distinct sites by calmodulin-dependent and cyclic-AMP-dependent kinases.

Microtubule-associated protein 2 (MAP2) is an excellent substrate for both cyclic-AMP (cAMP)-dependent and Ca2+/calmodulin-dependent kinases. A recently purified cytosolic Ca2+/calmodulin-dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule-associated cAMP-dependent and calmodulin-dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two-dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule-associated cAMP-dependent kinase and five major peptide species phosphorylated by calmodulin-dependent kinase. All 11 of the cAMP-dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin-dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP-dependent and calmodulin-dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.

Animals↗

PubMed1992

Differential responses of the phosphorylation of ribosomal protein S6 to nerve growth factor and epidermal growth factor in PC12 cells.

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.

Amino Acids↗