Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gbetagamma-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gbetagamma with high affinity and inhibits the interaction of Gbetagamma with Galpha or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gbetagamma. In this study, we report that Pd was rapidly phosphorylated by Ca(2+)/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gbetagamma binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca(2+)/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivo decreases in Ca(2+) concentration blocked the interaction of Pd with 14-3-3, indicating that Ca(2+) controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca(2+)-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.     

Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.     

Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells.

We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells.     

Cyclin-dependent kinase 11p110 and casein kinase 2 (CK2) inhibit the interaction between tyrosine hydroxylase and 14-3-3

Tyrosine hydroxylase (TH) is regulated by the reversible phosphorylation of serines 8, 19, 31 and 40. Upon initiation of this study, serine 19 was unique due to its requirement of 14-3-3 binding after phosphorylation for optimal enzyme activity, although it has been more recently demonstrated that phosphorylated serine 40 also binds 14-3-3. To identify proteins that interact with TH following phosphorylation of serine 19, this amino acid was mutated to alanine and THS19A was used as bait in a yeast two-hybrid system. From this, mouse-derived cyclin-dependent kinase 11 (CDK11)p110 was identified as an interacting partner with THS19A. The interaction was confirmed using human CDK11p110 cDNA in a mammalian system. Previous research has demonstrated that casein kinase 2 (CK2) interacts with CDK11p110, and both were observed to phosphorylate TH in vitro. In addition, CDK11p110 overexpression was observed to inhibit the interaction between TH and 14-3-3. A mechanism contributing to disruption of the interaction between TH and 14-3-3 may be due to CK2 phosphorylation of specific 14-3-3 isoforms, i.e. 14-3-3 tau. Collectively, these results imply that CDK11p110 and CK2 negatively regulate TH catecholamine biosynthetic activity since phosphoserine 19 of TH requires 14-3-3 binding for optimal enzyme activity and a decreased rate of dephosphorylation.     

Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.     

The 14-3-3 protein affects the conformation of the regulatory domain of human tyrosine hydroxylase

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases. Phosphorylation of Ser19 does not by itself increase TH activity but induces its binding to the 14-3-3 protein. That leads to the enhancement of TH activity with a still not fully understood mechanism. The main goal of this work was to investigate whether the 14-3-3 protein binding affects the conformation of the regulatory domain of human TH isoform 1 (TH1R). Site-directed mutagenesis was used to generate five single-tryptophan mutants of TH1R with the Trp residue located at five different positions within the domain (positions 14, 34, 73, 103, and 131). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser19 and Ser40 does not itself induce any significant structural changes in regions surrounding inserted tryptophans. On the other hand, the interaction between the 14-3-3 protein and phosphorylated TH1R decreases the solvent exposure of tryptophan residues at positions 14 and 34 and induces distinct structural change in the vicinity of Trp73. The 14-3-3 protein binding also reduces the sensitivity of phosphorylated TH1R to proteolysis by protecting its N-terminal part (first 33 residues). Circular dichroism measurements showed that TH1R is an unstructured protein with a low content of secondary structure and that neither phosphorylation nor the 14-3-3 protein binding changes its secondary structure.     

Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.     

Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A(2)

Calcium-/calmodulin-dependent protein kinase II (CaM kinase II), a decoder of Ca(2+) signals, and cytosolic phospholipase A(2) (cPLA(2)), an enzyme involved in arachidonate release, are involved in many physiological and pathophysiological processes. Activation of CaM kinase II in norepinephrine-stimulated vascular smooth muscle cells leads to activation of cPLA(2) and arachidonic acid release. Surface plasmon resonance, mass spectrometry, and kinetic studies show that CaM kinase II binds to cPLA(2) resulting in cPLA(2) phosphorylation on Ser-515 and an increase in its enzymatic activity. Phosphopeptide mapping studies with cPLA(2) from norepinephrine-stimulated smooth muscle cells indicates that phosphorylation of cPLA(2) on Ser-515, but not on Ser-505 or Ser-727, occurs in vivo. This novel signaling pathway for arachidonate release is shown to be cPLA(2)-dependent by use of a recently described and highly selective inhibitor of this enzyme.     

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.     

Site-specific phosphorylation of tyrosine hydroxylase after KCl depolarization and nerve growth factor treatment of PC12 cells

The phosphorylation and activation of tyrosine hydroxylase was examined in PC12 cells following depolarization with KCl or treatment with nerve growth factor. Both treatments activate tyrosine hydroxylase (TH) and increase enzyme phosphorylation. Site-specific analysis of the tryptic phosphopeptides of TH isolated from [32P]phosphate-labeled PC12 cells demonstrated that the major phosphorylated peptide (termed "H25") did not contain any of the previously reported phosphorylation sites. Phosphoamino acid analysis of this peptide demonstrated that the phosphorylated residue was a serine. Synthetic tryptic peptides containing putative phosphorylation sites were prepared, and subjected to high performance liquid chromatography analysis and isoelectric focusing. The tryptic phosphopeptide containing serine 31 comigrated with the H25 peptide during both of these analytical techniques. The tryptic phosphopeptide produced by the phosphorylation of tyrosine hydroxylase by the recently discovered proline-directed protein kinase and the phosphorylated synthetic phosphopeptide TH2-12 are clearly separated from H25 by this analysis. We conclude that serine 31 is phosphorylated during KCl depolarization and nerve growth factor treatment of PC12 cells and that this phosphorylation is responsible for the activation of tyrosine hydroxylase. Since this site is not located in a sequence selective for any of the "classical" protein kinases, we suggest that a novel protein kinase may be responsible for the phosphorylation of this site. Since serine 31 has a proline residue on the carboxyl-terminal side, the possibility that this kinase may be related to the recently reported proline-directed protein kinase is discussed. Other sites that are also phosphorylated on TH during KCl depolarization include serine 19, which is known to be phosphorylated by calmodulin-dependent protein kinase II. A schematic model for the regulation of tyrosine hydroxylase activity by phosphorylation of the NH2-terminal regulatory domain is presented.     

Site-specific phosphorylation of phosducin in intact retina. Dynamics of phosphorylation and effects on G protein beta gamma dimer binding

Phosducin (Pdc) is a G protein beta gamma dimer (G beta gamma) binding protein, highly expressed in retinal photoreceptor and pineal cells, yet whose physiological role remains elusive. Light controls the phosphorylation of Pdc in a cAMP and Ca(2+)-dependent manner, and phosphorylation in turn regulates the binding of Pdc to G(t)beta gamma or 14-3-3 proteins in vitro. To directly examine the phosphorylation of Pdc in intact retina, we prepared antibodies specific to the three principal phosphorylation sites (Ser-54, Ser-73, and Ser-106) and measured the kinetics of phosphorylation/dephosphorylation during light/dark adaptation and the subsequent effects on G(t)beta gamma binding. Ser-54 phosphorylation increased slowly (t((1/2)) approximately 90 min) during dark adaptation to approximately 70% phosphorylated and decreased rapidly (t((1/2)) approximately 2 min) during light adaptation to less than 20% phosphorylated. Ser-73 phosphorylation increased much faster during dark adaptation (t((1/2)) approximately 3 min) to approximately 50% phosphorylated and decreased more slowly during light adaptation (t((1/2)) approximately 9 min) to less than 20% phosphorylated. The Ca(2+) chelator BAPTA-AM blocked Ser-54 phosphorylation during dark adaptation but had no effect on Ser-73 phosphorylation. In contrast, Ser-106 was not phosphorylated in either the light or dark. Importantly, G beta gamma binding to Pdc was enhanced by Ca(2+) chelation and the binding kinetics closely paralleled those of Ser-54 dephosphorylation, indicating that Ser-54 phosphorylation controls G(t)beta gamma binding in vivo. These results suggest a pivotal role of Ser-54 and Ser-73 phosphorylation in determining the interactions of Pdc with its binding partners, G(t)beta gamma and 14-3-3 protein, which may regulate the light-dependent translocation of the photoreceptor G protein.     

Protein kinase A phosphorylation of human phosphodiesterase 3B promotes 14-3-3 protein binding and inhibits phosphatase-catalyzed inactivation

Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B.     

Serum and growth factors rapidly elicit phosphorylation of the Ca2+/calmodulin-dependent protein kinase II in intact quiescent rat 3Y1 cells

A 50-kDa protein was recognized in rat embryo fibroblast 3Y1 cells with an affinity-purified antibody against rat brain Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). When the cytosolic extract from quiescent 3Y1 cells was immunoprecipitated with the antibody, the 50-kDa protein in the immunoprecipitate became phosphorylated in a Ca2+- and calmodulin-dependent manner following exposure to [gamma-32P]ATP. Moreover, the reaction proceeded through an intramolecular mechanism. These results suggest that the 50-kDa protein is a subunit of CaM kinase II in rat 3Y1 cells. The addition of 10% fetal calf serum to quiescent 3Y1 cells caused a rapid increase in the phosphorylation of the 50-kDa protein, which was immunoprecipitated with the affinity-purified anti-CaM kinase II antibody. The phosphorylation of CaM kinase II was detected as early as 20 s after the addition of serum, reached the maximal level at 2 min, and decreased to the basal level within 60 min. Platelet-derived growth factor and epidermal growth factor also elicited the phosphorylation of the 50-kDa protein in quiescent 3Y1 cells, while neither insulin nor 12-O-tetradecanoylphorbol-13-acetate did. Calcium ionophores, A23187 and ionomycin, also caused the phosphorylation of the protein in 3Y1 cells. Moreover, phosphopeptide mappings of the phosphorylated 50-kDa subunit generated in response to serum, EGF, and A23187 yielded patterns similar to that generated from the immunoprecipitated 50-kDa subunit phosphorylated in vitro. Phosphoamino acid analysis of the phosphorylated subunit demonstrated that serine residue was the major amino acid labeled under any condition. These results suggest that CaM kinase II undergoes phosphorylation in response to various stimuli that can increase the free Ca2+ concentration in the cytoplasm of quiescent fibroblast cells and therefore probably mediates at least some of the biological actions of growth factors.     

Phosphorylation of microtubule-associated protein-2 in GH3 cells. Regulation by cAMP and by calcium.

The rat pituitary cell line GH3 contains a high molecular weight microtubule-associated protein with properties characteristic of microtubule-associated protein-2 (MAP-2). The 280-kDa protein is selectively immunoprecipitated by antibodies to authentic bovine brain MAP-2 and is phosphorylated at appropriate sites by cAMP-dependent protein kinase (cAMP kinase) and multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Although MAP-2 is a minor cellular constituent, it can be immunoprecipitated from [32P]Pi-labeled GH3 cells and shown to contain a high level of basal phosphorylation. Vasoactive intestinal peptide, forskolin, 3-isobutyl-1-methylxanthene, or cholera toxin, treatments which increase cellular cAMP levels, or dibutyryl cAMP stimulate phosphorylation of specific sites on MAP-2 without significantly increasing its high state of basal phosphorylation. Phosphopeptide mapping reveals that the sites phosphorylated by cAMP kinase in vitro are the same sites whose phosphorylation in situ increases following stimulation of GH3 with agents that activate cAMP kinase. Increasing intracellular Ca2+ levels in GH3 cells also stimulates phosphorylation of MAP-2 but at sites distinct from those phosphorylated following treatment with cAMP inducing agonists. Phosphopeptide mapping indicates that the sites phosphorylated by CaM kinase in vitro are the same sites whose phosphorylation in situ increases following Ca2(+)-mediated stimulation. We conclude that activation of cAMP- and Ca2(+)-based signaling pathways leads to phosphorylation of MAP-2 in GH3 cells and that cAMP kinase and CaM kinase mediate phosphorylation by these pathways, respectively.     

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca(2+)/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser(19), but PKA also phosphorylated Ser(104), as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser(19), Ser(99), Ser(104) and Ser(306). On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser(19). This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins gamma, epsilon and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.     

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.     

Identification of 14-3-3zeta as a protein kinase B/Akt substrate

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.     

L-triiodothyronine differentially and nongenomically regulates synaptosomal protein phosphorylation in adult rat brain cerebral cortex: role of calcium and calmodulin

Adult-onset thyroid disorders in humans impair several important central nervous system functions, causing various neuropsychiatric diseases. However, the mechanisms of thyroid hormone (TH) action in the mature mammalian brain remain unclear. Recent nongenomic actions of TH in adult brains are spotlighted. Many nongenomic mechanisms are modulated by phosphorylation-dephosphorylation of substrate proteins. In the present study, L-triiodothyronine (L-T3) demonstrated differential regulation of phosphorylation status of five different synaptosomal proteins (63, 53, 38, 23, and 16 kD) in both a Ca(2+)/calmodulin (CaM)-dependent and -independent manner. L-T3 increased the level of phosphorylation of all these five proteins. Ca(2+)/CaM further stimulated phosphorylation of 63- and 53-kD proteins by L-T3, which were inhibited both by EGTA (Ca(2+)-chelator) or KN62 (Ca(2+)/CaM kinase-II [CaMK-II] inhibitor), suggesting the role of CaMK-II. L-T3 increased the phosphorylation of 23- and 38-kD proteins; the effect was independent of EGTA or KN62. The presence of Ca(2+) decreased L-T3-induced phosphorylation of 63-, 53- and 38-kD proteins. Surprisingly, l-T3-induced phosphorylation of 16-kD protein was not augmented further with Ca(2+) or Ca(2+)/CaM; instead, the presence of CaM abolished the L-T3-induced phosphorylation. EGTA or KN62 could not restore the effect of CaM-induced dephosphorylation of this protein. This study identified the role of Ca(2+)/CaM in the regulation of L-T3-induced protein phosphorylation and supported a unique nongenomic mechanism of second messenger-mediated regulation of protein phosphorylation by TH in mature rat brain. This has profound implications for higher mental functions and strategies for novel therapeutics.