Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (ktr) in a length-dependent fashion

Force and power in cardiac muscle have a known dependence on phosphorylation of the myosin-associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force (ktr ) in maximally activating [Ca2+ ]. Activation was achieved by rapidly increasing the temperature (temperature-jump of 0.5-20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85-1.94 μm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC-exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates ktr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows ktr by ∼50%, relative to the ktr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of ktr following step shortening or ramp shortening. Using a two-state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross-bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca2+ ] and are not explained by changes in the Ca2+ -sensitivity of acto-myosin interactions. The length-dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank-Starling law of the heart.Published versio

Agrin-induced acetylcholine receptor clustering in mammalian muscle requires tyrosine phosphorylation.

Agrin is thought to be the nerve-derived factor that initiates acetylcholine receptor (AChR) clustering at the developing neuromuscularjunction. We have investigated the signaling pathway in mouse C2 myotubes and report that agrin induces a rapid but transient tyrosine phosphorylation of the AChR beta subunit. As the beta-subunit tyrosine phosphorylation occurs before the formation of AChR clusters, it may serve as a precursor step in the clustering mechanism. Consistent with this, we observed that tyrosine phosphorylation of the beta subunit correlated precisely with the presence or absence of clustering under several experimental conditions. Moreover, two tyrosine kinase inhibitors, herbimycin and staurosporine, that blocked beta-subunit phosphorylation also blocked agrin-induced clustering. Surprisingly, the inhibitors also dispersed preformed AChR clusters, suggesting that the tyrosine phosphorylation of other proteins may be required for the maintenance of receptor clusters. These findings indicate that in mammalian muscle, agrin-induced AChR clustering occurs through a mechanism that requires tyrosine phosphorylation and may involve tyrosine phosphorylation of the AChR itself

Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry

Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses – to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets

Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in <em>Saccharomyces cerevisiae</em>

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism

Erythropoietin-induced serine 727 phosphorylation of STAT3 in erythroid cells is mediated by a MEK-, ERK-, and MSK1-dependent pathway

Objective. Erythropoietin (EPO) is a key regulator of erythropoiesis, playing a role in both the proliferation and differentiation of erythroid cells. One of the signal transduction molecules activated upon EPO stimulation is signal transducer and activator of transcription (STAT) 3. Besides tyrosine 705 phosphorylation of STAT3, serine 727 phosphorylation has been described upon EPO stimulation. In the present study, we investigated which molecular pathways mediate the STAT3 serine 727 phosphorylation and the functional implications of this phosphorylation. Methods. The EPO-dependent erythroid cell line ASE2 was used to investigate which signaling routes were involved in the STAT3 serine 727 phosphorylation. Western blotting using phosphospecific antibodies was used to assess the phosphorylation status of STAT3 molecules. Transfection analysis was performed to investigate the transactivational potential of STAT3, and quantitative RT-PCR was used to study the in vivo gene expression of STAT3-responsive genes. Results. Western blotting of extracts of cells exposed to various chemical inhibitors revealed that the MEK inhibitors PD98059 and U0126 abrogated the EPO-mediated STAT3 serine 727 phosphorylation without an effect on tyrosine phosphorylation. Further analysis showed that MSK1 is activated downstream of ERK, and retroviral transductions with kinase-inactive MSK1 revealed that MSK1 is necessary for STAT3 serine phosphorylation. Furthermore, the STAT3-mediated transactivation was reduced by blocking the STAT3 serine phosphorylation with the MEK inhibitor U0126 or by expression of kinase-inactive MSK1. Conclusions. The EPO-induced STAT3 serine 727 phosphorylation is mediated by a pathway involving MEK, ERK, and MSK1. Furthermore, serine phosphorylation of STAT3 augments the transactivational potential of STAT3.

Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter.

The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against a number of pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whether PAMP-induced phosphorylation of PEN3 is important for its defense function or focal recruitment has not been addressed. In this study, we evaluated the role of PEN3 phosphorylation in modulating the localization and defense function of the transporter. We report that PEN3 phosphorylation is critical for its function in defense, but dispensable for recruitment to powdery mildew penetration sites. These results indicate that PAMP-induced phosphorylation is likely to regulate the transport activity of PEN3

Tau phosphorylation at Alzheimer\u27s disease-related Ser356 contributes to tau stabilization when PAR-1/MARK activity is elevated.

Abnormal phosphorylation of the microtubule-associated protein tau is observed in many neurodegenerative diseases, including Alzheimer\u27s disease (AD). AD-related phosphorylation of two tau residues, Ser262 and Ser356, by PAR-1/MARK stabilizes tau in the initial phase of mismetabolism, leading to subsequent phosphorylation events, accumulation, and toxicity. However, the relative contribution of phosphorylation at each of these sites to tau stabilization has not yet been elucidated. In a Drosophila model of human tau toxicity, we found that tau was phosphorylated at Ser262, but not at Ser356, and that blocking Ser262 phosphorylation decreased total tau levels. By contrast, when PAR-1 was co-overexpressed with tau, tau was hyperphosphorylated at both Ser262 and Ser356. Under these conditions, the protein levels of tau were significantly elevated, and prevention of tau phosphorylation at both residues was necessary to completely suppress this elevation. These results suggest that tau phosphorylation at Ser262 plays the predominant role in tau stabilization when PAR-1/MARK activity is normal, whereas Ser356 phosphorylation begins to contribute to this process when PAR-1/MARK activity is abnormally elevated, as in diseased brains

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase.

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation

Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel

Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, Naᵥ1.5. We show that Naᵥ1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. Naᵥ1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation–phosphorylation cross-talk of a cardiac ion channel

Phospho.ELM:a database of experimentally verified phosphorylation sites in eukaryotic proteins

BACKGROUND: Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a general need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins.DESCRIPTION: Phospho.ELM http://phospho.elm.eu.org is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed as part of the ELM (Eukaryotic Linear Motif) resource. Phospho.ELM constitutes the largest searchable collection of phosphorylation sites available to the research community. The Phospho.ELM entries store information about substrate proteins with the exact positions of residues known to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho.ELM version 2.0 contains 1703 phosphorylation site instances for 556 phosphorylated proteins.CONCLUSION: Phospho.ELM will be a valuable tool both for molecular biologists working on protein phosphorylation sites and for bioinformaticians developing computational predictions on the specificity of phosphorylation reactions.</p