The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.     

Regulation of endothelial nitric oxide synthase by protein kinase C

Endothelial nitric oxide synthase (eNOS) is a key enzyme in nitric oxide-mediated signal transduction in mammalian cells. Its catalytic activity is regulated both by regulatory proteins, such as calmodulin and caveolin, and by a variety of post-translational modifications including phosphorylation and acylation. We have previously shown that the calmodulin-binding domain peptide is a good substrate for protein kinase C [Matsubara, M., Titani, K., and Taniguchi, H. (1996) Biochemistry 35, 14651-14658]. Here we report that bovine eNOS protein is phosphorylated at Thr497 in the calmodulin-binding domain by PKC both in vitro and in vivo, and that the phosphorylation negatively regulates eNOS activity. A specific antibody that recognizes only the phosphorylated form of the enzyme was raised against a synthetic phosphopeptide corresponding to the phosphorylated domain. The antibody recognized eNOS immunoprecipitated with anti-eNOS antibody from the soluble fraction of bovine aortic endothelial cells, and the immunoreactivity increased markedly when the cells were treated with phorbol 12-myristate 13-acetate. PKC phosphorylated eNOS specifically at Thr497 with a concomitant decrease in the NOS activity. Furthermore, the phosphorylated eNOS showed reduced affinity to calmodulin. Therefore, PKC regulates eNOS activity by changing the binding of calmodulin, an eNOS activator, to the enzyme.     

Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells

Autophosphorylation of the insulin receptor was studied using a glycoprotein fraction solubilized and purified partially from the rat hepatoma cell line, Fao. Incubation of this receptor preparation with [gamma-32P] ATP, Mn2+, and insulin yielded a single insulin-stimulated phosphoprotein of Mr = 95,000 which corresponds to the beta-subunit of the insulin receptor. At 22 degrees C, incorporation of 32P was half-maximal at 30 s and about 90% complete after 2 min. At steady state, about 200 pmol of 32P were incorporated per mg of protein; this value corresponded to about 2 molecules of phosphate per insulin binding site estimated from Scatchard plots. Insulin increased the Vmax for autophosphorylation of the insulin receptor kinase nearly 20-fold with no effect on the Km for ATP. Mn2+ stimulated autophosphorylation by decreasing the Km of the kinase for ATP, whereas Mg2+ had no effect. Dilution of the insulin receptor over a 10-fold concentration range did not decrease the rate of autophosphorylation suggesting that it may occur by an intramolecular mechanism. When the phosphorylated beta-subunit of the insulin receptor was digested with trypsin, at least 5 phosphopeptides could be separated by high performance liquid chromatography on a mu Bondapak C18 reverse-phase column. Insulin stimulated the phosphorylation of all sites. These phosphate acceptor sites varied in their rate and degree of phosphorylation. Phosphopeptides pp4 and pp5 were phosphorylated very rapidly and reached steady state within 20 s, whereas phosphorylation of pp1 and pp2 required several minutes to reach steady state.     

Toward a hypothesis-free understanding of how phosphorylation dynamically impacts protein turnover

The turnover measurement of proteins and proteoforms has been largely facilitated by workflows coupling metabolic labeling with mass spectrometry (MS), including dynamic stable isotope labeling by amino acids in cell culture (dynamic SILAC) or pulsed SILAC (pSILAC). Very recent studies including ours have integrated themeasurement of post-translational modifications (PTMs) at the proteome level (i.e., phosphoproteomics) with pSILAC experiments in steady state systems, exploring the link between PTMs and turnover at the proteome-scale. An open question in the field is how to exactly interpret these complex datasets in a biological perspective. Here, we present a novel pSILAC phosphoproteomic dataset which was obtained during a dynamic process of cell starvation using data-independent acquisition MS (DIA-MS). To provide an unbiased “hypothesis-free” analysis framework, we developed a strategy to interrogate how phosphorylation dynamically impacts protein turnover across the time series data. With this strategy, we discovered a complex relationship between phosphorylation and protein turnover that was previously underexplored. Our results further revealed a link between phosphorylation stoichiometry with the turnover of phosphorylated peptidoforms. Moreover, our results suggested that phosphoproteomic turnover diversity cannot directly explain the abundance regulation of phosphorylation during cell starvation, underscoring the importance of future studies addressing PTM site-resolved protein turnover.     

Evidence against insulin-stimulated phosphorylation of calmodulin in 3T3-L1 adipocytes

To examine the possibility that insulin might stimulate calmodulin phosphorylation in intact cells, we compared autoradiographs of two-dimensional gels of [35S]methionine- and 32P-labeled proteins from 3T3-L1 adipocytes, before and after immunoprecipitation with anti-calmodulin antiserum. Insulin stimulated the phosphorylation of one or two proteins of approximately 22 kDa and pI 4.6; this increased phosphorylation was accompanied by an apparent shift in the position of the analogous [35S]methionine-labeled proteins towards the anode. In contrast, insulin had no effect on the phosphorylation state of another protein of 18-22 kDa and pI 4.6. This protein was very heavily labeled with [35S]methionine, co-migrated exactly with purified calmodulin, reacted specifically with two anti-calmodulin antibodies by Western blotting, and was specifically immunoprecipitated with the anti-calmodulin antiserum. Similar amounts of [35S]methionine-labeled calmodulin were immunoprecipitated from control and insulin-stimulated cells, arguing against the possibility that insulin-stimulated phosphorylation of calmodulin changed its affinity for the antibody. We conclude that calmodulin is phosphorylated to a negligible extent in serum-deprived 3T3-L1 adipocytes and that insulin does not stimulate its phosphorylation under conditions in which it stimulates the phosphorylation of one or more neighboring proteins.     

Antagonistic effects of insulin and beta-adrenergic agonists on the activity of protein phosphatase inhibitor-1 in skeletal muscle of the perfused rat hemicorpus

The extent of phosphorylation of protein phosphatase inhibitor-1 in skeletal muscle rose about 2.5-fold during 60 min of perfusion of the rat hemicorpus preparation and then did not change over the following 30 min. Addition of insulin at 60 min resulted in a 35% fall in inhibitor-1 phosphorylation by 90 min. The rise in inhibitor-1 phosphorylation was due to the presence of catecholamines as evidenced by an accumulation of epinephrine in the perfusate. Removal of the adrenal glands or cannulation of the vena cava prevented the accumulation of epinephrine and the rise in inhibitor-1 phosphorylation. Insulin did not alter the phosphorylation state of inhibitor-1 in the presence of the beta-adrenergic antagonist propranolol where the degree of phosphorylation was low (less than 10%) or at concentrations of isoproterenol (10 nM) where inhibitor-1 was highly phosphorylated (greater than 60%). In preparations with the adrenal glands removed, 0.5 nM isoproterenol produced a 2-fold rise in inhibitor-1 phosphorylation, an effect that was completely prevented by the addition of insulin. The antagonism of 0.5 nM idoproterenol by insulin correlated with a decrease in the muscle content of cyclic AMP. These results suggest that the dephosphorylation of inhibitor-1 may play an important role in the metabolic effects of insulin in vivo.     

Modifications of human histone H3 variants during mitosis

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.     

组氨酸磷酸化修饰的鉴定方法及应用

蛋白质磷酸化是广受关注的翻译后修饰类型之一,组氨酸磷酸化作为一种非常见的磷酸化修饰,最早被发现在细菌和低等真核生物信号传导的级联反应中起关键作用.近年来研究显示,其在肿瘤发生发展过程中也可能扮演了重要角色.由于磷酸化组氨酸的化学不稳定性、低丰度、亚化学计量性质、缺乏特异性的富集试剂,导致研究手段缺乏,限制了人们对磷酸化...     

Insulin stimulates the phosphorylation of calmodulin in intact adipocytes

Phosphorylation of cellular proteins is known to play an important role in mediating the metabolic effects of insulin in target cells. Here we show that exposure of intact adipocytes to physiological concentrations of insulin results in phosphorylation of the calcium receptor protein, calmodulin. The identity of the phosphorylated protein as being calmodulin in intact cells was demonstrated by two-dimensional electrophoresis, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7)-affinity chromatography, and positive staining with the Ca2+ binding protein stain Stains All. Phosphorylation of calmodulin occurred at physiological insulin concentrations with maximum stimulation (608 +/- 114% over basal) at 50 microunits/ml (3.3 X 10(-10) M) insulin. The 32Pi incorporated into calmodulin was stable to base, indicating that phosphotyrosine was involved and thus implicating the insulin-receptor tyrosine kinase as being responsible for its phosphorylation. The phosphorylation of calmodulin may represent an important component of the mechanism for intracellular signaling not only for insulin, but potentially for other physiological regulators of cellular metabolism.     

The insulin receptor and calmodulin. Calmodulin enhances insulin-mediated receptor kinase activity and insulin stimulates phosphorylation of calmodulin

Despite intensive research efforts, the functional role and regulation of the insulin receptor kinase remain enigmatic. In this investigation, we demonstrate that calmodulin enhances insulin-stimulated phosphorylation of the beta subunit of the insulin receptor and histone H2b and that insulin also stimulates phosphorylation of calmodulin. Using wheat germ lectin-enriched insulin receptor preparations obtained from rat adipocyte plasma membranes, calmodulin stimulated the rate and increased the amount of 32P incorporated predominantly into tyrosine residues of the beta subunit of the receptor when assayed in the presence of insulin. The stimulatory effect of calmodulin was both dose-dependent and saturable with half-maximal and maximal phosphorylation of the beta subunit occurring at 0.4 and 2.0 microM calmodulin, respectively. Ca2+ enhanced the ability of calmodulin to stimulate insulin-mediated phosphorylation of the beta subunit with an apparent K0.5 of approximately 0.6 microM. Calmodulin also induced an approximately 2-fold increase in both the rate and amount of insulin-mediated incorporation of 32P into histone H2b. The stimulatory effect of calmodulin was only observed in the presence of insulin and was concentration-dependent (K0.5 approximately 3.0 microM calmodulin), saturable (at 5 microM calmodulin), and Ca2+-dependent (K0.5 = 0.2 microM free Ca2+). Insulin also induced phosphorylation of a 17-kDa protein. On the basis of its molecular weight and purification via immunoadsorption with protein A-Sepharose-bound anti-calmodulin IgG, this phosphoprotein was identified as a phosphorylated form of calmodulin. Phosphorylation of calmodulin was only observed in the presence of insulin and was both Ca2+- and insulin concentration-dependent with half-maximal effects observed at 0.1 microM free Ca2+ and 350 microunits/ml insulin. Collectively, these results support the hypothesis that Ca2+ and calmodulin participate in the molecular mechanism whereby binding of insulin to its receptor is coupled to changes in cellular metabolism.     

A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin

Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.     

Tyrosine phosphorylation of the insulin receptor during insulin-stimulated internalization in rat hepatoma cells

We have studied the phosphorylation state of the insulin receptor during receptor-mediated endocytosis in the well-differentiated rat hepatoma cell line Fao. Insulin induced the rapid internalization of surface-iodinated insulin receptors into a trypsin-resistant compartment, with a 3-fold increase in the internalization rate over that seen in the absence of insulin. Within 20 min of insulin stimulation, 30-35% of surface receptors were located inside the cell. This redistribution was half-maximal by 10.5 min. Similar results were obtained when the loss of surface receptors was measured by 125I-insulin binding. Tyrosyl phosphorylation of internalized insulin receptors was measured by immunoprecipitation with antiphosphotyrosine antibody. Immediately after insulin stimulation, 70-80% of internalized receptors were tyrosine phosphorylated. Internalized receptors persisted in a phosphorylated state after the dissociation of insulin but were dephosphorylated prior to their return to the plasma membrane. After 45-60 min of insulin stimulation, the tyrosine phosphorylation of the internal receptor pool decreased by 45%, whereas the phosphorylation of surface receptors was unchanged. These data suggest that insulin induces the internalization of phosphorylated insulin receptors into the cell and that the phosphorylation state of the internal receptor pool may be regulated by insulin.     

In vivo phosphorylation of human erythrocyte spectrin occurs in a sequential manner

Spectrin is the major component of the erythrocyte membrane skeleton and exists as a 526 kDa alphabeta heterodimer. The 246 kDa beta-chain of human spectrin is phosphorylated near the C-terminus, but the exact phosphorylation sites are unknown and the role of this phosphorylation is not fully characterized. In this study, we produced a monoclonal antibody, Sp316, capable of recognizing the C-terminal region of beta-spectrin regardless of its phosphorylation state and used it to purify the phosphorylated region after 2-nitro-5-thiocyanobenzoic acid cleavage of spectrin. Two-dimensional gels, mass spectrometry, and reversed-phase high-performance liquid chromatography were used to characterize these phosphorylation states. Only about 1.5% of spectrin isolated from fresh blood is unphosphorylated, about 9% has more than four phosphates per molecule, and the majority of the protein has one to four phosphates per molecule. A total of six phosphorylation sites were identified by tandem mass spectrometry. Quantitative analysis of the phosphorylation states by reversed-phase high-performance liquid chromatography revealed that phosphorylation of beta-spectrin occurs in a sequential manner where each specific site is completely phosphorylated before the next site is modified. The first phosphorylation event occurs on Ser-2114, followed by Ser-2125, Ser-2123, Ser-2128, Ser-2117, and Thr-2110. The identification of the specific phosphorylated beta-spectrin residues and the ordered sequence of phosphorylation events in vivo should provide an invaluable basis for further studies of the role of these posttranslational modifications in spectrin function in situ.     

Analysis of the in vivo phosphorylation state of protein phosphatase inhibitor-2 from rabbit skeletal muscle by fast-atom bombardment mass spectrometry

A new procedure has been developed for identifying phosphoserine residues in proteins, and is used to analyse the in vivo phosphorylation state of inhibitor-2. The method employs reverse-phase liquid chromatography to resolve phosphorylated and dephosphorylated forms of peptides and fast-atom bombardment mass spectrometry (FABMS) to identify phosphorylated derivatives. The positions of phosphorylation sites within peptides are located by gas-phase sequencer analysis after conversion of phosphoserine residues to S-ethylcysteine. The phosphorylation sites on inhibitor-2 were identified as serines-86, -120 and -121, the three residues phosphorylated in vitro by casein kinase-II. Serine-86 was phosphorylated to 0.7 mol/mol and serines-120 and -121 each to 0.3 mol/mol. These values were not altered significantly by intravenous injection of adrenalin or insulin. No phosphate was present in the region comprising residues 1-49, even after injection of adrenalin, demonstrating that inhibitor-2 is not a substrate for cyclic AMP-dependent protein kinase in vivo. The absence of phosphotyrosine also indicated that inhibitor-2 is not a physiological substrate for the insulin receptor. Surprisingly, no phosphate was present at threonine-72, the residue phosphorylated in vitro by glycogen synthase kinase-3, after injection of either propranolol, adrenalin or insulin. The implications of this finding for the in vivo activation of protein phosphatase 1I (the 1:1 complex between inhibitor-2 and the catalytic subunit of protein phosphatase-1) are discussed. FABMS analysis of inhibitor-2 confirmed the accuracy of the primary structure reported previously, and showed that the only post-translational modifications were an N-acetyl moiety and the three phosphoserine residues. FABMS also demonstrated the presence of an additional serine residue at the C-terminus, and showed that 50% of isolated inhibitor-2 molecules lack the C-terminal Ser-Ser dipeptide.     

Engineering mammalian mucin-type O-glycosylation in plants

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.     

The phototrophic bacterium Rhodopseudomonas capsulata sp108 encodes an indigenous class A beta-lactamase.

It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin receptor in a Ca2+-dependent manner, whereas it binds to polylysine seemingly by electrostatic interactions. These studies identify calmodulin as a substrate for the highly purified insulin receptor tyrosine kinase of human placenta. They also demonstrate that the basic proteins, which are required for insulin to stimulate the phosphorylation of calmodulin, do so by a direct interaction with calmodulin.     

Evidence for exogenous substrate phosphorylation and dimer formation by the activated 190 kDa insulin proreceptor in vitro

The insulin receptor is synthesized as a single chain, 190 kDa glycoprotein precursor, which undergoes proteolytic cleavage, carbohydrate processing, and fatty acylation to generate the mature receptor on the plasma membrane. The relationship of these post-translational modifications to the acquisition of receptor function, i.e. ligand binding and phosphokinase activity, is not fully understood. Therefore, the 190 kDa proreceptor and mature receptor kinase activities were separately examined in vitro, and their phosphorylation properties compared. The solubilized receptor precursor from IM-9 lymphocytes was purified by sequential lectin chromatography and, following site specific anti-receptor antibody immunoprecipitation, phosphokinase studies performed. The isolated proreceptor was activated by insulin and phosphorylated exogenous substrate alpha-casein, as similarly observed for the mature receptor. Structurally, the phosphorylated proreceptor was identified as a 360 kDa homodimer under non-reducing condition.