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Apoptosis-associated tyrosine kinase 1 (AATYK1), a novel serine/threonine kinase that is highly expressed in the brain, is involved in neurite extension and apoptosis of cerebellar granule neurons; however, its precise function remains unknown. In this study, we investigated the interaction of AATYK1A with Cyclin-dependent kinase 5 (Cdk5)/p35, a proline-directed protein kinase that is predominantly expressed in neurons. AATYK1A bound to the p35 activation subunit of Cdk5 in cultured cells and in mouse brains and colocalized with p35 on endosomes in COS-7 cells. AATYK1A was phosphorylated at Ser34 by Cdk5/p35 in vitro, in cultured neurons and in mouse brain. In PC12D cells, Ser34 phosphorylation increased after treatment with nerve growth factor and phosphorylated AATYK1A accumulated in growth cones of PC12D cells. Ser34 phosphorylation suppressed the tyrosine phosphorylation of AATYK1A by Src family kinases. These results suggest a possibility that AATYK1A plays a role in early to recycling endosomes and its function is regulated by phosphorylation with Cdk5 or Src-family kinases.
Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at approximately 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.
In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2ACdc55 phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2ACdc55 specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control.
Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100-200nM) to regulate the phosphorylation of large quantities of PLB (50μM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100-200 fold lower concentrations.
Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome-wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high-confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine-specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho-residues in Arabidopsis is similar to that in humans, where over 90 tyrosine-specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.
STAT2 is an essential transcription factor in type I IFN mediated anti-viral and anti-proliferative signaling. STAT2 function is regulated by tyrosine phosphorylation, which is the trigger for STAT-dimerization, subsequent nuclear translocation, and transcriptional activation of IFN stimulated genes. Evidence of additional STAT2 phosphorylation sites has emerged as well as novel roles for STAT2 separate from the classical ISGF3-signaling. This review aims to summarize knowledge of phosphorylation-mediated STAT2-regulation and future avenues of related STAT2 research.
During the Hsp90-mediated chaperoning of protein kinases, the core components of the machinery, Hsp90 and the cochaperone Cdc37, recycle between different phosphorylation states that regulate progression of the chaperone cycle. We show that Cdc37 phosphorylation at Y298 results in partial unfolding of the C-terminal domain and the population of folding intermediates. Unfolding facilitates Hsp90 phosphorylation at Y197 by unmasking a phosphopeptide sequence, which serves as a docking site to recruit non-receptor tyrosine kinases to the chaperone complex via their SH2 domains. In turn, Hsp90 phosphorylation at Y197 specifically regulates its interaction with Cdc37 and thus affects the chaperoning of only protein kinase clients. In summary, we find that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner.
DNA dependent Protein Kinase (DNA-PK) is an heterotrimeric complex regulating the Non Homologous End Joining (NHEJ) double strand break (DSB) repair pathway. The activity of its catalytic subunit (DNA-PKcs) is regulated by multiple phosphorylations, like the Ser2056 one that impacts DSB end processing and telomeres integrity. O-GlcNAcylation is a post translational modification (PTM) closely related to phosphorylation and its implication in the modulation of DNA-PKcs activity during the DNA Damage Response (DDR) is unknown.
The goals of this study were to examine the cellular signaling pathways associated with the phosphorylation of caldesmon, the phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17), and the 20-kDa regulatory light chain of myosin (MLC20) induced by levobupivacaine in isolated rat aortas. The effects of genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, 1-butanol, and ML-7 HCl on levobupivacaine-induced contraction were assessed. The effect of genistein on the simultaneous calcium-tension curves induced by levobupivacaine was examined. The effects of GF109203X, genistein, PD98059 and extracellular signal-regulated kinase (ERK) siRNA on levobupivacaine-induced caldesmon phosphorylation were investigated. The effect of genistein on the ERK and tyrosine phosphorylation induced by levobupivacaine was examined. The effect of GF109203X, PD98059, Y-27632, SP600125, and ML-7 HCl on the levobupivacaine-induced phosphorylation of CPI-17 and MLC20 were investigated. Genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, ML-7 HCl, and 1-butanol attenuated levobupivacaine-induced contraction. Genistein caused a right downward shift of the calcium-tension curves induced by levobupivacaine. Genistein attenuated levobupivacaine-induced phosphorylation of protein tyrosine, ERK and caldesmon. PD98059, ERK siRNA and GF109203X attenuated levobupivacaine-induced caldesmon phosphorylation. GF109203X, Y-27632, SP600125, ML-7 HCl and PD98059 attenuated CPI-17 phosphorylation and MLC20 phosphorylation induced by levobupivacaine. These results suggest that levobupivacaine-induced caldesmon phosphorylation contributing to levobupivacaine-induced contraction is mediated by a pathway involving ERK, which is activated by tyrosine kinase or protein kinase C (PKC). The phosphorylation of CPI-17 and MLC20 induced by levobupivacaine is mediated by cellular signaling pathways involving PKC, Rho-kinase, and c-Jun NH2-terminal kinase or PKC, Rho-kinase, ERK, and myosin light chain kinase.
SIR2 is an NAD(+)-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported.
Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied.
The androgen receptor (AR) has been identified for decades and mediates essential steroid functions. Like most of biological molecules, AR functional activities are modulated by post-translational modifications. This review is focused on the reported activities and significance of AR phosphorylation, with particular emphasis on proline-directed serine/threonine phosphorylation that occurs predominantly on the receptor. The marked enrichment of AR phosphorylation in the most diverse N-terminal domain suggests that targeting AR phosphorylation can be synergistic to antagonizing the C-terminal domain by clinical antiandrogens.
The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic function of PS1/γ-secretase can be modulated by its 'phosphorylated' and 'dephosphorylated' states. However, the functional outcome of PS1 phosphorylation and its significance for Alzheimer's disease (AD) pathogenesis is poorly understood. Here, comprehensive analysis using FRET-based imaging reveals that activity-driven and Protein Kinase A-mediated PS1 phosphorylation at three domains (domain 1: T74, domain 2: S310 and S313, domain 3: S365, S366, and S367), with S367 being critical, is responsible for the PS1 pathogenic 'closed' conformation, and resulting increase in the Aβ42/40 ratio. Moreover, we have established novel imaging assays for monitoring PS1 conformation in vivo, and report that PS1 phosphorylation induces the pathogenic conformational shift in the living mouse brain. These phosphorylation sites represent potential new targets for AD treatment.
Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5-ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5⁻/⁻ cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation.
Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.
The quantitative model of cyclin-dependent kinase (CDK) function states that cyclins temporally order cell cycle events at different CDK activity levels, or thresholds. The model lacks a mechanistic explanation, as it is not understood how different thresholds are encoded into substrates. We show that a multisite phosphorylation code governs the phosphorylation of CDK targets and that phosphorylation clusters act as timing tags that trigger specific events at different CDK thresholds. Using phospho-degradable CDK threshold sensors with rationally encoded phosphorylation patterns, we were able to predictably program thresholds over the entire range of the Saccharomyces cerevisiae cell cycle. We defined three levels of CDK multisite phosphorylation encoding: (i) serine-threonine swapping in phosphorylation sites, (ii) patterning of phosphorylation sites, and (iii) cyclin-specific docking combined with modulation of CDK activity. Thus, CDK can signal via hundreds of differentially encoded targets at precise times to provide a temporally ordered phosphorylation pattern required for cell division.
In eukaryotes, mRNA translation is dependent on the cap-binding protein eIF4E. Through its simultaneous interaction with the mRNA cap structure and with the ribosome-associated eIF4G adaptor protein, eIF4E physically posits the ribosome at the 5' extremity of capped mRNA. eIF4E activity is regulated by phosphorylation on a unique site by the eIF4G-associated kinase MNK. eIF4E assembly with the eIF4G-MNK sub-complex can be however antagonized by the hypophosphorylated forms of eIF4E-binding protein (4E-BP). We show here that eIF4E phosphorylation is dramatically affected by disruption of eIF4E-eIF4G interaction, independently of changes in MNK expression. eIF4E phosphorylation is actually strongly downregulated upon eIF4G shutdown or upon sequestration by hypophosphorylated 4E-BP, consequent to mTOR inhibition. Downregulation of 4E-BP renders eIF4E phosphorylation insensitive to mTOR inhibition. These data highlight the important role of 4E-BP in regulating eIF4E phosphorylation independently of changes in MNK expression.
Two isoforms of Rho-associated protein kinase (ROCK), ROCKI and ROCKII, play a pivotal role in regulation of cytoskeleton and are involved in multiple cellular processes in mammalian cells. Knockout mice experiments have indicated that the functions of ROCKI and II are probably non-redundant in physiology. However, it is difficult to differentiate the activation status of ROCKI and ROCKII in biological samples. Previously, we have identified phosphorylation site of ROCKII at Ser1366 residue sensitive to ROCK inhibition. We further investigated the activity-dependent phosphorylation site in ROCKI to establish the reagents that can be used to detect their individual activation.
In Alzheimer's disease (AD) and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A) activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of neurofibrillary pathology.
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