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PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1's role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.

Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.

We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1. We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site. To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine. We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF. These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases.

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.

Hyperphosphorylated high molecular weight neurofilament protein (NF-H) exhibits extensive phosphorylation on lysine-serine-proline (KSP) repeats in the C-terminal domain of the molecule. Specific phosphorylation sites in human NF-H were identified by proteolytic digestion and analysis of the resulting digests by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry, and database searching. The computer programs utilized (PEPSEARCH and SEQUEST) are capable of identifying peptides and phosphorylation sites from uninterpreted MS/MS spectra, and by use of these methods, 27 phosphopeptides and their phosphorylated residues were identified. On the basis of these phosphopeptides, 38 phosphorylation sites in human NF-H were characterized. These include 33 KSP, lysine-threonine-proline (KTP) or arginine-serine-proline (RSP) sites and four unphosphorylated sites, all of which occur in the KSP repeat domain (residues 502-823); and one threonine phosphorylation site observed in a KVPTPEK motif. Six KSP sites were not characterized because of the failure to isolate and identify corresponding phosphopeptides. Heterogeneity in serine and threonine phosphorylation was observed at three sites or deduced to occur at three sites on the basis of enzyme specificity. As a result of the phosphorylated motifs identified (KSPAKEE, KSPVKEE, KS/TPEKAK, KSPEKEE, KSPVKAE, KSPAEAK, KSPPEAK, KSPEAKT, KSPAEVK, and KVPTPEK), human NF-H tail domain is postulated to be a substrate of proline-directed kinases. The threonine-phosphorylated KVPTPEK motif suggested the existence of a novel proline-directed kinase.

The distribution of light energy between the two photosystems as well as the light-induced turnover of PSII proteins are regulated by the reversible phosphorylation of LHCII and the PSII-core proteins. The thylakoid protein kinase(s) is activated by a signal transduction system involving the interaction of reduced plastoquinone with the quinol oxidation site of the cytochrome bf complex [1]. Phosphorylation of the mobile pool of LHCII induces dissociation of this antenna from PSII and allows its interaction with the PSI in the stroma exposed membranes (state transition)[21. Dephosphorylation of LHCII by a membrane -bound phosphatase appears to be regulated by a cyclophilinlike protein located in the thylakoid lumen [1, 31. So far, it was assumed that the role of light in the above processes is only to drive the photosynthetic electron flow thus reducing plastoquinone and triggering the signal transduction system resulting in the protein kinase(s) activation. In this work we have used an in vitro reconstituted system consisting of partially purified, solubilized thylakoid protein-kinase(s) and isolated native as well as recombinant LHCII. We demonstrate that light induces conformational changes in the LHCII structure exposing the specific threonine phosphorylation site to the kinase activity, thus suggesting a regulation of protein phosphorylation also at the substrate level.

Phosphorylation of prolactin by endogenous protein kinases within isolated secretory granules was shown to result in the production of both phosphoserine and phosphothreonine residues. The majority of the radiolabel was determined to be present in the C terminus of the molecule after specific cleavage with glandular kallikrein. Glandular kallikrein cleaves in three places at the C terminus, liberating three small peptides, only one of which contains a phosphorylatable residue. Sequencing of this phosphopeptide showed it to be Arg175-Lys185. Thus the major site of prolactin phosphorylation was determined to be serine 177. Using a synthetic peptide equivalent to this region of the molecule (Ser161-Val180), serine 177 was demonstrated to be a substrate for protein kinase A as well as for one of the endogenous granule kinases. Inclusion of the synthetic peptide in an endogenous granule phosphorylation reaction resulted in competition for the kinase and reduced phosphorylation of prolactin. Protein kinase A phosphorylation of purified prolactin resulted in the production of only phosphoserine and primarily the most abundant (monophosphorylated) variant. We conclude that serine 177 is the major in vivo phosphorylation site of rat prolactin and that phosphorylation of this site can be reproduced by protein kinase A in vitro. The minor threonine phosphorylation site was demonstrated by two-dimensional tryptic peptide mapping and mass analysis to be either threonine 58 or 63, both of which are contained within a single peptide.

The protein CD36 is a membrane receptor for thrombospondin (TSP), malaria-infected erythrocytes, and collagen. Three functional sequences were identified within a single disulfide loop of CD36: one that mediates TSP binding (amino acids 87 to 99) and two that support malarial cytoadhesion (amino acids 8 to 21 and 97 to 110). One of these peptides (p87-99) is a consensus protein kinase C (PKC) phosphorylation site. Dephosphorylation of constitutively phosphorylated CD36 in resting platelets and a megakaryocytic cell line led to the loss of collagen adhesion and platelet reactivity to collagen, with a reciprocal increase in TSP binding. PKC-mediated phosphorylation of this ectodomain resulted in a loss of TSP binding and the reciprocal acquisition of collagen binding. In site-directed mutagenesis studies, when the threonine phosphorylation site was changed to alanine, CD36 was expressed in a dephosphorylated state and bound to TSP constitutively.

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.

Main conclusion Phosphorylation and dephosphorylation events were initiated in wheat scab resistance. The putative FHB-responsive phosphoproteins are mainly involved in three functional groups and contain at least one tyrosine, serine, or threonine phosphorylation site. Fusarium head blight (FHB), caused by Fusarium graminearum, is a severe disease in wheat. Protein phosphorylation plays an important role in plant–pathogen interactions, however, a global analysis of protein phosphorylation in response to FHB infection remains to be explored. To study the effect of FHB on the phosphorylation state of wheat proteins, proteins extracted from spikes of a resistant wheat cultivar after 6 h of inoculation with F. graminearum or sterile H2O were separated by two-dimensional gel electrophoresis, and then the immunodetection of putative phosphoproteins was conducted by Western blotting using specific anti-phosphotyrosine antibody, anti-phosphothreonine antibody and anti-phosphoserine antibody. A total of 35 phosphorylated signals was detected and protein identities of 28 spots were determined. Functional categorization showed that the putative FHB-responsive phosphoproteins were mainly involved in defense/stress response, signal transduction, and metabolism. The phosphorylation status of proteins associated with signaling pathways mediated by salicylic acid, calcium ions, small GTPase, as well as with detoxification, reactive oxygen species scavenging, antimicrobial compound synthesis, and cell wall fortification was regulated in wheat spikes in response to F. graminearum infection. The present study reveals dynamics of wheat phosphoproteome in response to F. graminearum infection and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of wheat scab resistance. Electronic supplementary material The online version of this article (doi:10.1007/s00425-015-2441-y) contains supplementary material, which is available to authorized users.