Genetic Encoding of Phosphorylated Amino Acids into Proteins.

Reversible phosphorylation is a fundamental mechanism for controlling protein function. Despite the critical roles phosphorylated proteins play in physiology and disease, our ability to study individual phospho-proteoforms has been hindered by a lack of versatile methods to efficiently generate homogeneous proteins with site-specific phosphoamino acids or with functional mimics that are resistant to phosphatases. Genetic code expansion (GCE) is emerging as a transformative approach to tackle this challenge, allowing direct incorporation of phosphoamino acids into proteins during translation in response to amber stop codons. This genetic programming of phospho-protein synthesis eliminates the reliance on kinase-based or chemical semisynthesis approaches, making it broadly applicable to diverse phospho-proteoforms. In this comprehensive review, we provide a brief introduction to GCE and trace the development of existing GCE technologies for installing phosphoserine, phosphothreonine, phosphotyrosine, and their mimics, discussing both their advantages as well as their limitations. While some of the technologies are still early in their development, others are already robust enough to greatly expand the range of biologically relevant questions that can be addressed. We highlight new discoveries enabled by these GCE approaches, provide practical considerations for the application of technologies by non-GCE experts, and also identify avenues ripe for further development.

The RCC1 family protein SAB1 negatively regulates ABI5 through multidimensional mechanisms during postgermination in Arabidopsis.

Abscisic acid-insensitive 5 (ABI5) is an essential and conserved plant basic leucine zipper transcription factor whose level controls seed germination and postgerminative development. It has been demonstrated that activity of ABI5 is transcriptionally and post-translationally regulated. However, transcriptional regulation of ABI5 is not fully understood. Here, we identified SAB1 (Sensitive to ABA 1) as a novel negative regulator of ABI5 that simultaneously regulates its stability, promoter binding activity and histone methylation-mediated gene silencing of ABI5. SAB1 encodes a Regulator of Chromatin Condensation 1 (RCC1) family protein and is expressed in an opposite pattern to that of ABI5 during early seedling growth in response to abscisic acid (ABA). SAB1 mutation results in enhanced ABA sensitivity and acts upstream of ABI5. SAB1 physically interacts with ABI5 at phosphoamino acid Ser-145, and reduces the phosphorylation of ABI5 and the protein stability. SAB1 reduces ABI5 binding activity to its own promoter, leading to reduced transcriptional level of ABI5. SAB1 inactivates ABI5 transcription by increasing the level of histone H3K27me2 in the ABI5 promoter. Our findings have identified SAB1 as a crucial new component of ABA signaling which modulates early development of plant by precisely controlling ABI5 activity through multiple mechanisms.

Phosphatase-Stable Phosphoamino Acid Mimetics That Enhance Binding Affinities with the Polo-Box Domain of Polo-like Kinase†1.

(2S,3R)-2-Amino-3-methyl-4-phosphonobutanoic acid (Pmab) is a phosphatase-stable analogue of phosphothreonine (pThr), which has been used in a variety of biological contexts. Among these applications are peptidomimetic ligands that bind to the polo-box domain (PBD) of polo-like kinase 1 (Plk1) with affinities approaching that of the corresponding pThr-containing peptides. However, Pmab is not widely used, because there are no direct, high-yield preparations of suitably protected reagent. We have now achieved an efficient synthesis of protected Pmab, as well as variants with different substituents at the 3R center. When incorporated into our peptidomimetic scaffold, these new Pmab analogues exhibit Plk1 PBD-binding affinities that are several-fold higher than Pmab, yet retain good selectivity for Plk1 relative to the PBDs of Plk2 and Plk3. These findings will significantly impact the future development of PBD-binding inhibitors, as well as ligands directed against a broad spectrum of pThr-dependent processes.

Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer.

Altered protein phosphorylation is a feature of many human cancers that can be targeted therapeutically. Phosphopeptide enrichment is a critical step for maximizing the depth of phosphoproteome coverage by MS, but remains challenging for tissue specimens because of their high complexity. We describe the first analysis of a tissue phosphoproteome using polymer-based metal ion affinity capture (PolyMAC), a nanopolymer that has excellent yield and specificity for phosphopeptide enrichment, on a transgenic mouse model of HER2-driven breast cancer. By combining phosphotyrosine immunoprecipitation with PolyMAC, 411 unique peptides with 139 phosphotyrosine, 45 phosphoserine, and 29 phosphothreonine sites were identified from five LC-MS/MS runs. Combining reverse phase liquid chromatography fractionation at pH 8.0 with PolyMAC identified 1571 unique peptides with 1279 phosphoserine, 213 phosphothreonine, and 21 phosphotyrosine sites from eight LC-MS/MS runs. Linear motif analysis indicated that many of the phosphosites correspond to well-known phosphorylation motifs. Analysis of the tyrosine phosphoproteome with the Drug Gene Interaction database uncovered a network of potential therapeutic targets centered on Src family kinases with inhibitors that are either FDA-approved or in clinical development. These results demonstrate that PolyMAC is well suited for phosphoproteomic analysis of tissue specimens.

TNNI3K, a cardiac-specific kinase, promotes physiological cardiac hypertrophy in transgenic mice.

PURPOSE: Protein kinase plays an essential role in controlling cardiac growth and hypertrophic remodeling. The cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac specific kinase, is associated with cardiomyocyte hypertrophy. However, the precise function of TNNI3K in regulating cardiac remodeling has remained controversial. METHODS AND RESULTS: In a rat model of cardiac hypertrophy generated by transverse aortic constriction, myocardial TNNI3K expression was significantly increased by 1.62 folds (P<0.05) after constriction for 15 days. To investigate the role of TNNI3K in cardiac hypertrophy, we generated transgenic mouse lines with overexpression of human TNNI3K specifically in the heart. At the age of 3 months, the high-copy-number TNNI3K transgenic mice demonstrated a phenotype of concentric hypertrophy with increased heart weight normalized to body weight (1.31 fold, P<0.01). Echocardiography and non-invasive hemodynamic assessments showed enhanced cardiac function. No necrosis or myocyte disarray was observed in the heart of TNNI3K transgenic mice. This concentric hypertrophy maintained up to 12 months of age without cardiac dysfunction. The phospho amino acid analysis revealed that TNNI3K is a protein-tyrosine kinase. The yeast two-hybrid screen and co-immunoprecipitation assay identified cTnI as a target for TNNI3K. Moreover, TNNI3K overexpression induced cTnI phosphorylation at Ser22/Ser23 in vivo and in vitro, suggesting that TNNI3K is a novel upstream regulator for cTnI phosphorylation. CONCLUSION: TNNI3K promotes a concentric hypertrophy with enhancement of cardiac function via regulating the phosphorylation of cTnI. TNNI3K could be a potential therapeutic target for preventing from heart failure.

Analysis of Janus tyrosine kinase phosphorylation and activation.

Activation of Janus kinases (Jaks) occurs through autophosphorylation of key tyrosine residues located primarily within their catalytic domain. Phosphorylation of these tyrosine residues facilitates access of substrates to the active site and serves as an intrinsic indicator of Jak activation. Here, we describe the methods and strategies used for analyzing Jak phosphorylation and activation. Tyrosine-phosphorylated (active) Jaks are primarily detected from cell extracts using anti-phosphotyrosine-directed Western blot analysis of Jak-specific immunoprecipitates. Additionally, receptor pull-down and in vitro kinase assays can also be utilized to measure cellular Jak catalytic activity. In addition to tyrosine phosphorylation, recent evidence indicates Jaks can be serine phosphorylated upon cytokine stimulation, however the lack of commercially available antibodies to detect these sites has hindered their analysis by Western blot. However, phosphoamino acid analysis (PAA) has been employed to monitor Jak serine and threonine phosphorylation. Over the past decade, remarkable advances have been made in our understanding of Jak function and dysfunction, however much remains to be learned about their complex regulatory mechanisms.

Effects on polo-like kinase 1 polo-box domain binding affinities of peptides incurred by structural variation at the phosphoamino acid position.

Protein-protein interactions (PPIs) mediated by the polo-box domain (PBD) of polo-like kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr ß-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.

P-N bond protein phosphatases.

The current work briefly reviews what is currently known about protein phosphorylation on arginine, lysine and histidine residues, where PN bonds are formed, and the protein kinases that catalyze these reactions. Relatively little is understood about protein arginine and lysine kinases and the role of phosphorylation of these residues in cellular systems. Protein histidine phosphorylation and the two-component histidine kinases play important roles in cellular signaling systems in bacteria, plants and fungi. Their roles in vertebrates are much less well researched and there are no protein kinases similar to the two-component histidine kinases. The main focus of the review however, is to present current knowledge of the characterization, mechanisms of action and biological roles of the phosphatases that catalyze the hydrolysis of these phosphoamino acids. Very little is known about protein phosphoarginine and phospholysine phosphatases, although their existence is well documented. Some of these phosphatases exhibit very broad specificity in terms of which phosphoamino acids are substrates, however there appear to be one or two quite specific protein phospholysine and phosphoarginine phosphatases. Similarly, there are phosphatases with broad substrate specificities that catalyze the hydrolysis of phosphohistidine in protein substrates, including the serine/threonine phosphatases 1, 2A and 2C. However there are two, more specific, protein phosphohistidine phosphatases that have been well characterized and for which structures are available, SixA is a phosphatase associated with two-component histidine kinase signaling in bacteria, and the other is found in a number of organisms, including mammals. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

Proteomic screening method for phosphopeptide motif binding proteins using peptide libraries.

Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC-MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp, or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were applied to columns containing these phosphorylated and nonphosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested, and analyzed by LC-MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy/light peptide ion ratios were calculated, and peptides that yielded ratios greater than ∼3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro, and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected, and to a lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2, and PH domains; however, pSer/pThr motifs did not reveal enriched domains across hits.

Sequential activation and deactivation of protein function using spectrally differentiated caged phosphoamino acids.

Photolabile caging groups, including the 1-(2-nitrophenyl)ethyl (NPE) group, have been applied to probe many biological processes, including protein phosphorylation. Although studies with NPE-caged phosphoamino acids have provided valuable information, these investigations have been limited to the use of only one caged species in a single experiment. To expand the scope of these tools, we have developed an approach for sequentially uncaging two different phosphopeptides in one system, enabling interrogation of multiple phosphorylation events. We present the synthesis of [7-(diethylamino)coumarin-4-yl]methyl (DEACM)-caged phosphorylated serine, threonine, and tyrosine building blocks for Fmoc-based solid-phase peptide synthesis to allow convenient incorporation of these residues into peptides and proteins. Exposure of DEACM- and NPE-caged phosphopeptides to 420 nm light selectively releases the DEACM group without affecting the NPE-caged peptide. This then enables a subsequent irradiation event at 365 nm to remove the NPE group and liberate a second phosphopeptide. We demonstrate the versatility of this general sequential uncaging approach by applying it to control Wip1 phosphatase with two wavelengths of light.

Survey of activated FLT3 signaling in leukemia.

Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.

Protein kinase CK2 regulates the formation and clearance of aggresomes in response to stress.

Misfolded protein aggregates elicit a stress response, and their clearance is crucial for cell survival. These aggregates are transported by cytoplasmic deacetylase HDAC6 and dynein motors to the aggresome via the microtubule network, and are removed by autophagic degradation. HDAC6 activity is necessary for both the transport and clearance of protein aggregates. However, the cellular factors that regulate HDAC6 activity remain unknown. Here we show that protein kinase CK2 is a crucial modulator of HDAC6 activity because CK2 directly phosphorylates HDAC6 and increases cytoplasmic deacetylase activity. Indeed, cells that expressed HDAC6 mutated at Ser458, a CK2-mediated phosphorylation site, failed to both form and clear aggresomes, and increased cytotoxicity. Interestingly, Ser458 is conserved only in higher primates, such as human and chimpanzee, but not in the rhesus macaque. These findings identify CK2 as a crucial protein involved in the formation and clearance of aggresomes, and hence in cell viability in response to misfolded protein stress.

Luminescent kinase activity biosensors based on a versatile bimolecular switch.

Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a fluorescence resonance energy transfer (FRET)-based readout, has often been a limiting factor. Here we show that a kinase-inducible bimolecular switch consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility. Specifically, we demonstrate that bimolecular switches designed to sense protein kinase A (PKA) or protein kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges in comparison with their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

BACKGROUND: The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs. PRINCIPAL FINDINGS: PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+)-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr(137), Thr(141), Thr(174) and Thr(290) being the target residues of PknB while Thr(137) and Thr(174) are phosphorylated by PknA. The Mn(2+)-ion binding residues Asp(38) and Asp(229) are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c) (D38G) are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+)/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent. CONCLUSIONS AND SIGNIFICANCE: This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

Stabilization of amorphous calcium carbonate by phosphate rich organic matrix proteins and by single phosphoamino acids.

Stable amorphous calcium carbonate (ACC) is a unique material produced naturally exclusively as a biomineral. It was demonstrated that proteins extracted from biogenic stable ACC induce and stabilize synthetic ACC in vitro. Polyphosphate molecules were similarly shown to induce amorphous calcium carbonate formation in vitro. Accordingly, we tested the hypothesis that biogenic ACC induction and stabilization is mediated by the phosphorylated residues of phosphoproteins. We show that extracellular organic matrix extracted from gastroliths of the red claw crayfish Cherax quadricarinatus induce stable ACC formation in vitro. The proteinaceous fraction of this organic matrix is highly phosphorylated and is incorporated into the ACC mineral phase during precipitation. We have identified the major phosphoproteins of the organic matrix and showed that they have high calcium binding capacity. Based on the above, in vitro precipitation experiments with single phosphoamino acids were performed, indicating that phosphoserine or phosphothreonine alone can induce the formation of highly stable ACC. The results indicate that phosphoproteins may play a major role in the control of ACC formation and stabilization and that their phosphoamino acid moieties are key components in this process.

Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species.

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole-genome sequencing projects reporting the presence of serine/threonine kinases and two-component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in-depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non-enteric Gram-negative bacteria captured by a nanoLC-MS-based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.

Regulation of cytosolic phospholipase A2alpha by hsp90 and a p54 kinase in okadaic acid-stimulated macrophages.

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA(2)alpha is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA(2)alpha-induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54-kDa kinase (p54), which phosphorylated cPLA(2)alpha in in-gel kinase assays and was associated with cPLA(2)alpha in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA(2)alpha. The results demonstrate a role for hsp90 in regulating cPLA(2)alpha-mediated AA release that involves association of a p54 kinase with cPLA(2)alpha upon OA stimulation.

Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana.

In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.

EmbR2, a structural homologue of EmbR, inhibits the Mycobacterium tuberculosis kinase/substrate pair PknH/EmbR.

EmbR is a transcriptional regulator that is phosphorylated by the cognate mycobacterial STPK (serine/threonine protein kinase) PknH. Recent studies demonstrated that PknH-dependent phosphorylation of EmbR enhances its DNA-binding activity and activates the transcription of the embCAB genes encoding arabinosyltransferases, which participate in arabinan biosynthesis. In the present study, we identified a genomic region of 4425 bp, which is present in Mycobacterium tuberculosis CDC1551, but absent from M. tuberculosis H37Rv, comprising the MT3428 gene, which is homologous with embR. Homology modelling of the MT3428 gene product illustrated its close relationship (56% identity) to EmbR, and it was hence termed EmbR2. In marked contrast with EmbR, EmbR2 was not phosphorylated by PknH, although it is a substrate of other M. tuberculosis kinases, including PknE and PknF. Tryptophan fluorescence emission of EmbR2 was monitored in the presence of three different PknH-derived phosphopeptides and demonstrated that EmbR2 binds to at least two of the threonine sites known to undergo autophosphorylation in PknH. We observed that the capacity of EmbR2 to interact physically with PknH without being phosphorylated was a result of EmbR2-mediated inhibition of kinase activity: incubation of PknH with increasing concentrations of EmbR2 led to a dose-response inhibition of the autokinase activity, similarly to O6-cyclohexylmethylguanine, a known inhibitor of eukaryotic cyclin-dependent kinases. Moreover, EmbR2 inhibited PknH-dependent phosphorylation of EmbR in a dose-dependent manner. Together, these results suggest that EmbR2 is a regulator of PknH activation, thus directly participating in the control of the PknH/EmbR pair and potentially in mycobacterial physiology/virulence of M. tuberculosis CDC1551.

Biochemical characterization of phospholipids, sulfatide and heparin as potent stimulators for autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein in vitro.

The stimulatory effects of SH (sulfatide and heparin) and two phospholipids (PI and PS) on autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein (MBP) and two synthetic MBP peptides (M86 and M156) were comparatively examined in vitro. It was found that (i) both PI and SH highly stimulated the GSK-3beta-mediated phosphorylation of MBP, but not glycogen synthase, and two MBP peptides through their direct binding to these substrates and (ii) both PI and heparin, as compared with sulfatide, highly stimulated autophosphorylation of GSK-3beta. The K(m) value of MBP for GSK-3beta was highly reduced and the V(max) value was significantly increased in the presence of these acidic modulators, which augmented further phosphorylation of MBP by the kinase. Under our experimental condition, similar stimulatory effects of PI and heparin were observed with the GSK-3beta-mediated phosphorylation of tau protein (TP) in vitro. These results presented here suggest that these two phospholipids and SH may function as effective stimulators for autophosphorylation of GSK-3beta and for the GSK-3beta-mediated high phosphorylation of SH-binding proteins, including MBP and TP, in the highly accumulated levels of these acidic and sulfated modulators in the brain.