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PubMed1998

Serum and insulin induce a Grb2-dependent shift in agonist affinity of beta-adrenergic receptors.

Beta-adrenergic receptors transduce catecholamine binding to activation of adenylylcyclase, a response counter-regulated by insulin. Insulin stimulates tyrosine phosphorylation of Tyr 350/354, which abolishes the catecholamine response. Phosphorylation of Try 350/354 creates a Src homology 2 (SH2) domain on the beta2-adrenergic receptor and the binding of adaptor protein Grb2 to this SH2 domain of the beta-adrenergic receptor takes place in an insulin-dependent manner. In membranes from serum-deprived S49 mouse lymphoma cells, GTPgammaS yields the well-known agonist-specific shift in agonist affinity for beta2-adrenergic receptors. The agonist-specific shift is observed in cell membranes either in the absence or in the presence of exogenously added purified Grb2. In membranes for serum-fed cells, in contrast, the addition of Grb2 induces an agonist-specific shift in receptor affinity that mimics addition of GTPgammaS to the membranes. The ability of the Grb2 to induce an agonist-specific shift in the membranes from serum-fed cells was abolished equally effectively either by competition with phosphopeptide harbouring the (p)YVNV motif or by disruption of the SH2 domain of added Grb2. Challenging Chinese hamster ovary cells with insulin (100 nM) for 30 min enabled Grb2 to induce an agonist-specific shift in agonist affinity for beta2-adrenergic receptors, suggestive of uncoupling of the receptors from G proteins. The insulin-dependent Grb2 effect on receptor-G-protein coupling was sensitive to competition by the pYVNY phosphopeptide and to disruption of the SH2 domain of Grb2. These data provide a biochemical link between the ability of insulin to counter-regulate catecholamine stimulation of cyclic AMP accumulation and the phosphorylation of the beta-adrenergic receptor, consequent biding of the adaptor molecule Grb2 and disruption of receptor-G-protein coupling.

Adaptor Proteins, Signal Transducing↗

PubMed2003

pp60c-src activation in lung adenocarcinoma.

Nine src family members are known including c-Src, c-Yes, c-Lck, c-Fyn, c-Hck, c-Lyn, c-Blk, c-Fgr and c-Yrk. They encode proteins with molecular weights of 55-62 kilodaltons (kDa), which are either cytoplasmic or membrane-associated protein tyrosine kinases. A close correlation exists between an elevated pp60c-src tyrosine kinase activity and cell transformation. However, the level of activation of pp60c-src in non-small cell lung cancers (NSCLC) remains obscure. The aim of this study was to examine the level of activity of pp60c-src in NSCLC. pp60c-src expression and in vitro protein tyrosine kinase activity in lung cancer tissue samples were measured by western blotting and in vitro kinase assays and compared with those in the surrounding non-tumour lung tissue from the same patient. pp60c-src phosphorylation was assessed by two-dimensional tryptic phosphopeptide mapping. The kinase activity of pp60c-src was significantly activated in NSCLC, especially in adenocarcinomas. In addition, the pp60c-src kinase activity increased with the size of the adenocarcinoma. Two-dimensional tryptic phosphopeptide mapping showed dephosphorylation of pp60c-src at Tyr 530 in adenocarcinomas. The proto-oncogene product, pp60c-src, was activated in NSCLC, especially in adenocarcinomas, in part through the dephosphorylation of Tyr 530. Our results suggest that activation of pp60c-src might play an important role in the progression of lung adenocarcinomas.

Adenocarcinoma↗

PubMed2002

Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint.

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.

Binding Sites↗

PubMed2001

Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry.

Capillary liquid chromatography (muLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by muLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins beta-casein, beta-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by muLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides.

Amino Acid Sequence↗

PubMed2002

Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing.

Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.

Amino Acid Sequence↗

PubMed2004

Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites.

Protein phosphorylation is one of the most important known posttranslational modifications. Tandem mass spectrometry has become an important tool for mapping out the phosphorylation sites. However, when a peptide generated from the enzymatic or chemical digestion of a phosphoprotein is highly phosphorylated or contains many potential phosphorylation residues, phosphorylation site assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based site determination. In this work, we present a novel open tubular immobilized metal ion affinity chromatography (OT-IMAC) method, which is found to be more effective and reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptip from Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown that MALDI MS/MS with collision-induced dissociation can be very effective in generating fragment ion spectra containing rich structural information, which enables the identification of phosphorylation sites even from highly phosphorylated peptides. The applicability of this method for real world applications is demonstrated in the characterization and identification of phosphorylation sites of a Na(+)/H(+) exchanger fusion protein, His182, which was phosphorylated in vitro using the kinase Erk2.

Journal Article↗

PubMed2004

Analysis of phosphorylated peptides by ion mobility-mass spectrometry.

An ion mobility-mass spectrometry technique for rapid screening of phosphopeptides in protein digests is described. A data set of 43 sequences (ranging in mass from 400 to 3000 m/z) of model and tryptic peptides, including serine, threonine, and tyrosine phosphorylation, was investigated, and the data support our previously reported observation (Ruotolo, B. T.; Verbeck, G. F., IV; Thomson, L. M.; Woods, A. S.; Gillig, K. J.; Russell, D. H. J. Proteome Res. 2002, 1, 303.) that the drift time-m/z relationship for singly charged phosphorylated peptide ions is different from that for nonphosphorylated peptides. The data further illustrate that a combined data-dependent IM-MS/MS approach for phosphopeptide screening would have enhanced throughput over conventional MS/MS-based methodologies.

Amino Acid Sequence↗

PubMed2006

Strategy for the identification of sites of phosphorylation in proteins: neutral loss triggered electron capture dissociation.

We have previously demonstrated the suitability of data-dependent electron capture dissociation (ECD) for incorporation into proteomic strategies. The ability to directly determine sites of phosphorylation is a major advantage of electron capture dissociation; however, the low stoichiometry associated with phosphorylation means that phosphopeptides are often overlooked in data-dependent ECD analyses. In contrast, collision-induced dissociation (CID) tends to result in loss of the labile phosphate group, often at the expense of sequence fragments. Here, we demonstrate a novel strategy for the characterization of phosphoproteins which exploits the neutral loss feature of CID such that focused ECD of phosphopeptides is achieved. Peptides eluting from a liquid chromatograph are first subjected to CID, and if a neutral loss of 98 Da (corresponding to H3PO4) from the precursor is observed, ECD of that same precursor is performed; i.e., the method comprises neutral loss triggered ECD (NL-ECD-MS/MS). The method was applied to tryptic digests of beta-casein and alpha-casein. For alpha-casein, four sites of phosphorylation were identified with NL-ECD-MS/MS compared with a single site identified by ECD-MS/MS. The method also resulted in ECD of a doubly phosphorylated peptide. A further benefit of the method is that overall protein sequence coverage is improved. Sequence information from nonphosphorylated peptides is obtained as a result of the CID step.

Amino Acid Sequence↗

PubMed1993

Phosphorylation of human and bovine prothymosin alpha in vivo.

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.

Alkaline Phosphatase↗

PubMed2000

Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro.

Gab-1 (Grb2-associated binder-1), which appears to play a central role in cellular growth response, transformation, and apoptosis, is a member of the insulin receptor substrate (IRS) family. IRS proteins act downstream in the signaling pathways of different receptor tyrosine kinases, including the insulin receptor (IR). In this paper, we characterize the phosphorylation of recombinant human Gab-1 (hGab-1) by IR in vitro. Kinetic phosphorylation data revealed that hGab-1 is a high affinity substrate for the IR (K(M): 12.0 microM for native IR vs 23.3 microM for recombinant IR). To elucidate the IR-specific phosphorylation pattern of hGab-1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Phosphorylated tyrosine residues were subsequently identified by sequencing the separated phosphopeptides by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that hGab-1 was phosphorylated by IR at eight tyrosine residues (Y242, Y285, Y373, Y447, Y472, Y619, Y657, and Y689). Seventy-five percent of the identified radioactivity was incorporated into tyrosine residues Y447, Y472, and Y619 exhibiting features (NYVPM motif) of potential binding sites for the regulatory subunit (p85) of phosphatidylinositol (PI)-3 kinase. Accordingly, pull down assays with human HepG2 cell lysates showed that IR-specific phosphorylation of wild-type hGab-1 strongly enhanced PI-3 kinase binding. This is still the case when a single tyrosine residue in the NYVPM motif was mutated to phenylalanine. In contrast, phosphorylation-dependent binding of PI-3 kinase was completely abolished by changing a second tyrosine residue in a NYVPM motif independent from its location. Recently, we identified a similar cohort of tyrosine phosphorylation sites for the epidermal growth factor receptor (EGFR) with a predominant phosphorylation of tyrosine residue Y657 and binding of Syp [Lehr, S. et al. (1999) Biochemistry 38, 151-159]. These differences in the phosphorylation pattern of hGab-1 may contribute to signaling specificity by different tyrosine kinase receptors engaging distinct SH2 signaling molecules.

Adaptor Proteins, Signal Transducing↗

PubMed1994

GDP as a regulator of phosphorylation of elongation factor 1 by casein kinase II.

Elongation factor 1 (EF-1) consists of four subunits: the alpha subunit catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes while the beta, gamma, and delta subunits catalyze GDP/GTP exchange on EF-1 alpha. Phosphorylation of the beta subunit of EF-1 from rabbit reticulocytes by casein kinase II was stimulated up to 22-fold by polylysine, while basic proteins or polyarginine enhanced phosphorylation to a lesser extent. When physiological components of protein synthesis were examined as potential modulators of phosphorylation, ribosomal subunits had no effect, tRNA and poly(U) inhibited the phosphotransferase reaction, and GDP stimulated the initial rate of phosphorylation of EF-1 beta up to 3.8-fold; the degree of stimulation could be correlated with the amount of alpha subunit present in EF-1. No stimulation was observed with other nucleotides. Phosphorylation of EF-1 beta was on serine, and two-dimensional phosphopeptide mapping showed a single tryptic phosphopeptide in the presence of GDP or polylysine; the peptide was identical to that obtained with EF-1 phosphorylated in reticulocytes incubated with [32P]orthophosphate. EF-1 delta was also phosphorylated by casein kinase II, but only in the presence of GDP. Kinetic data showed GDP stimulated phosphorylation by increasing the Vmax with both the beta and delta subunits. The GDP-dependent stimulation of phosphorylation was specific for EF-1 and was not observed with calmodulin, beta-casein B, or c-Myc.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Tyrosine 508 of the 85-kilodalton subunit of phosphatidylinositol 3-kinase is phosphorylated by the platelet-derived growth factor receptor.

The mechanisms by which growth factors and oncogenic agents activate phosphatidylinositol 3-kinase (PI3 kinase) are unknown. Previously, we reported that the 85-kDa regulatory subunit of PI3 kinase is tyrosine-phosphorylated both in vitro by the platelet-derived growth factor beta-receptor (PDGFR) tyrosine kinase and in fibroblasts in response to PDGF. As a first step in determining the role of tyrosine phosphorylation in PDGF signaling through PI3 kinase, we investigated which tyrosines on p85 are phosphorylated by the PDGFR. Recombinant p85 was phosphorylated with recombinant PDGF receptors, and tryptic phosphopeptides were purified by HPLC and analyzed by Edman degradation. By this approach and by mutational analysis, Y508 was identified as the major in vitro phosphorylation site. Tryptic phosphopeptide mapping demonstrated Y508 to also be phosphorylated in vivo in COS cells. Comparison of these data with a previous report [Hayashi, H., Nishioka, Y., Kamohara, S., Kanai, F., Ishii, K., Fukui, Y., Shibasaki, F., Takenawa, T., Kido, H., Katsunuma, N., & Ebina, Y. (1993) J. Biol. Chem. 268, 7107-7117] suggests that p85 is phosphorylated differently by the PDGF and insulin receptor tyrosine kinases. Therefore, p85 may be regulated differently by PDGF and insulin. Mapping of phosphorylation sites on p85 may lead to new insights into the regulation of signal transduction through PI3 kinase.

Amino Acid Sequence↗

PubMed1989

Identification of phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent protein kinase on the voltage-sensitive sodium channel from Electrophorus electricus.

The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation.

Amino Acid Sequence↗

PubMed2004

In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

Amino Acid Sequence↗

PubMed2004

Identification of major ERK-related phosphorylation sites in Gab1.

Gab1 (Grb2-associated binder1) belongs to a family of multifunctional docking proteins that play a central role in the integration of receptor tyrosine kinase (RTK) signaling, i.e., mediating cellular growth response, transformation, and apoptosis. In addition to RTK-specific tyrosine phosphorylation, these docking proteins also can be phosphorylated on serine/threonine residues affecting signal transduction. Since serine and threonine phosphorylation are capable of modulating the initial signal one major task to elucidate signal transduction via Gab1 is to determine the exact localization of distinct phosphorylation sites. To address this question in this report we examined extracellular signal-regulated kinases 1/2 (ERK) specific serine/threonine phosphorylation of the entire Gab1 engaged in insulin signaling in more detail in vitro. To elucidate the ERK1/2-specific phosphorylation pattern of Gab1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Subsequently, phosphorylated serine/threonine residues were identified by sequencing the separated phosphopeptides using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that ERK1/2 phosphorylate Gab1 at six serine/threonine residues (T312, S381, S454, T476, S581, S597) in consensus motifs for MAP kinase phosphorylation. Serine residues S454, S581, S597, and threonine residue T476 represent nearly 80% of overall incorporated phosphate. These sites are located adjacent to src homology region-2 (SH2) binding motifs (YVPM-motif: Y447, Y472, Y619) specific for the phosphatidylinositol 3kinase (PI3K). The biological role of identified phosphorylation sites was proven by PI3K and Akt activity in intact cells. These data demonstrate that ERK1/2 modulate insulin action via Gab1 by targeting serine and threonine residues beside YXXM motifs. Accordingly, insulin signaling is blocked at the level of PI3K.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites.

Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.

Animals↗

PubMed1996

Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor.

The cDNA encoding human brain protein phosphatase inhibitor-1 (I-1) was expressed in Escherichia coli. Following PKA phosphorylation at a threonine, recombinant human I-1 was indistinguishable from rabbit skeletal muscle I-1 as a potent and specific inhibitor of the type-1 protein serine/threonine phosphatase (PP1). N-Terminal phosphopeptides of I-1 that retained the selectivity of intact human I-1 highlighted a functional domain that mediates PP1 inhibition. Substituting alanine in place of threonine-36 eliminated I-1 phosphorylation by PKA and its phosphatase inhibitor activity. An acidic residue was substituted in place of the phosphoacceptor to produce I-1(T35D), a constitutive phosphate inhibitor. I-1(T35D) was an equally effective inhibitor of PP1 and the type-2 phosphatase, PP2A. However, CNbr digestion of I-1(T35D) yielded an N-terminal peptide that showed 100-fold increased specificity as a PP1 inhibitor. This provided new insight into a unique conformation of the phosphorylated I-1 that accounts for selective inhibition of PP1 activity. Truncation of an active I-1 phosphopeptide identified an N-terminal sequence that was reduced in addition to threonine-35 phosphorylation to inhibit PP1 activity. Biosensor studies demonstrated that PP1 bound to both Phosphorylated and dephosphorylated I-1 and suggested that distinct elements of I-1 structure accounted for PP1 binding and inhibition. Our data point to multiple interactions between the I-1 functional domain. and the PP1 catalytic subunit that define this phosphoprotein as a physiological regulator of the type-1 protein phosphatase.

Amino Acid Sequence↗

PubMed1999

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins.

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.

14-3-3 Proteins↗