Consensus Phosphorylation Sites Research Articles

Salt-Inducible Kinase 1 Terminates cAMP Signaling by an Evolutionarily Conserved Negative-Feedback Loop in β-Cells.

Pancreatic β-cells are critical in the regulation of glucose homeostasis by controlled secretion of insulin in mammals. Activation of protein kinase A by cAMP is shown to be responsible for enhancing this pathway, which is countered by phosphodiesterase (PDE) that converts cAMP to AMP and turns off the signal. Salt-inducible kinases (SIKs) were also known to inhibit cAMP signaling, mostly by promoting inhibitory phosphorylation on CREB-regulated transcription coactivators. Here, we showed that SIK1 regulates insulin secretion in β-cells by modulating PDE4D and cAMP concentrations. Haploinsufficiency of SIK1 led to the improved glucose tolerance due to the increased glucose-stimulated insulin secretion. Depletion of SIK1 promoted higher cAMP concentration and increased insulin secretion from primary islets, suggesting that SIK1 controls insulin secretion through the regulation of cAMP signaling. By using a consensus phosphorylation site of SIK1, we identified PDE4D as a new substrate for this kinase family. In vitro kinase assay as well as mass spectrometry analysis revealed that the predicted Ser(136) and the adjacent Ser(141) of PDE4D are critical in SIK1-mediated phosphorylation. We found that overexpression of either SIK1 or PDE4D in β-cells reduced insulin secretion, while inhibition of PDE4 activity by rolipram or knockdown of PDE4D restored it, showing indeed that SIK1-dependent phosphorylation of PDE4D is critical in reducing cAMP concentration and insulin secretion from β-cells. Taken together, we propose that SIK1 serves as a part of a self-regulatory circuit to modulate insulin secretion from pancreatic β-cells by controlling cAMP concentration through modulation of PDE4D activity.

Phosphorylation of Sae2 Mediates Forkhead-associated (FHA) Domain-specific Interaction and Regulates Its DNA Repair Function

Saccharomyces cerevisiae Sae2 and its ortholog CtIP in higher eukaryotes have a conserved role in the initial processing of DNA lesions and influencing their subsequent repair pathways. Sae2 is phosphorylated by the ATR/ATM family kinases Mec1 and Tel1 in response to DNA damage. Among the Mec1/Tel1 consensus phosphorylation sites of Sae2, we found that mutations of Thr-90 and Thr-279 of Sae2 into alanine caused a persistent Rad53 activation in response to a transient DNA damage, similar to the loss of Sae2. To gain insight into the function of this phosphorylation of Sae2, we performed a quantitative proteomics analysis to identify its associated proteins. We found that phosphorylation of Thr-90 of Sae2 mediates its interaction with Rad53, Dun1, Xrs2, Dma1, and Dma2, whereas Rad53 and Dun1 additionally interact with phosphorylated Thr-279 of Sae2. Mutations of the ligand-binding residues of Forkhead-associated (FHA) domains of Rad53, Dun1, Xrs2, Dma1, and Dma2 abolished their interactions with Sae2, revealing the involvement of FHA-specific interactions. Mutations of Thr-90 and Thr-279 of Sae2 caused a synergistic defect when combined with sgs1Δ and exo1Δ and elevated gross chromosomal rearrangements. Likewise, mutations of RAD53 and DUN1 caused a synthetic growth defect with sgs1Δ and elevated gross chromosomal rearrangements. These findings suggest that threonine-specific phosphorylation of Sae2 by Mec1 and Tel1 contributes to DNA repair and genome maintenance via its interactions with Rad53 and Dun1.

Phosphorylation and Alternative Splicing of 7B2 Reduce Prohormone Convertase 2 Activation.

FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues within a variety of different bone proteins. In this work we demonstrate that FAM20C also phosphorylates threonines, specifically those within the N-terminal domain of the neuroendocrine chaperone 7B2. Analysis of the primary sequence of 7B2 revealed that three threonine residues in its N-terminal domain are located within FAM20C consensus motifs: Thr73, Thr99, and Thr111. The individual substitution of Thr73 and Thr111 residues by neutral alanines caused a marked decrease in the total phosphorylation of 7B2. Furthermore, the phosphomimetic substitution of Thr111 by Glu clearly diminished the ability of 7B2 to activate pro-prohormone convertase 2 (PC2) in 7B2-lacking SK-N-MC neuroblastoma cells, suggesting that the phosphorylation of this residue critically impacts the 7B2-proPC2 interaction. However, the phosphomimetic mutation did not alter 7B2's ability to function as an antiaggregant for human islet amyloid polypeptide. FAM20C-mediated phosphorylation of a common alternatively spliced variant of human 7B2 that lacks Ala100 (thus eliminating the Thr99 phosphorylation consensus site) was similar to the Ala-containing protein, but this variant did not activate proPC2 as efficiently as the Ala-containing protein. Although threonines within 7B2 were phosphorylated efficiently, FAM20C was incapable of performing the well-known regulatory threonine phosphorylation of the molecular chaperone binding immunoglobulin protein. Taken together, these results indicate that FAM20C plays a role in 7B2-mediated proPC2 activation by phosphorylating residue Thr111; and that 7B2 function is regulated by alternative splicing.

Loss of Ypk1, the Yeast Homolog to the Human Serum- and Glucocorticoid-induced Protein Kinase, Accelerates Phospholipase B1-mediated Phosphatidylcholine Deacylation

Ypk1, the yeast homolog of the human serum- and glucocorticoid-induced kinase (Sgk1), affects diverse cellular activities, including sphingolipid homeostasis. We now report that Ypk1 also impacts the turnover of the major phospholipid, phosphatidylcholine (PC). Pulse-chase radiolabeling reveals that a ypk1Δ mutant exhibits increased PC deacylation and glycerophosphocholine production compared with wild type yeast. Deletion of PLB1, a gene encoding a B-type phospholipase that hydrolyzes PC, in a ypk1Δ mutant curtails the increased PC deacylation. In contrast to previous data, we find that Plb1 resides in the ER and in the medium. Consistent with a link between Ypk1 and Plb1, the levels of both Plb1 protein and PLB1 message are elevated in a ypk1Δ strain compared with wild type yeast. Furthermore, deletion of PLB1 in a ypk1Δ mutant exacerbates phenotypes associated with loss of YPK1, including slowed growth and sensitivity to cell wall perturbation, suggesting that increased Plb1 activity buffers against the loss of Ypk1. Because Plb1 lacks a consensus phosphorylation site for Ypk1, we probed other processes under the control of Ypk1 that might be linked to PC turnover. Inhibition of sphingolipid biosynthesis by the drug myriocin or through utilization of a lcb1-100 mutant results in increased PLB1 expression. Furthermore, we discovered that the increase in PLB1 expression observed upon inhibition of sphingolipid synthesis or loss of Ypk1 is under the control of the Crz1 transcription factor. Taken together, these results suggest a functional interaction between Ypk1 and Plb1 in which altered sphingolipid metabolism up-regulates PLB1 expression via Crz1.

Protein interactions of the vesicular glutamate transporter VGLUT1.

Exocytotic release of glutamate depends upon loading of the neurotransmitter into synaptic vesicles by vesicular glutamate transporters, VGLUTs. The major isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in synapses of the adult rodent brain that correlates with the probability of release and potential for plasticity. Indeed, expression of different VGLUT protein isoforms confers different properties of release probability. Expression of VGLUT1 or 2 protein also determines the kinetics of synaptic vesicle recycling. To identify molecular determinants that may be related to reported differences in VGLUT trafficking and glutamate release properties, we investigated some of the intrinsic differences between the two isoforms. VGLUT1 and 2 exhibit a high degree of sequence homology, but differ in their N- and C-termini. While the C-termini of VGLUT1 and 2 share a dileucine-like trafficking motif and a proline-, glutamate-, serine-, and threonine-rich PEST domain, only VGLUT1 contains two polyproline domains and a phosphorylation consensus sequence in a region of acidic amino acids. The interaction of a VGLUT1 polyproline domain with the endocytic protein endophilin recruits VGLUT1 to a fast recycling pathway. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of in vitro biochemical screening assays using the region encompassing the polyproline motifs, phosphorylation consensus sites, and PEST domain. We identify interactors that belong to several classes of proteins that modulate cellular function, including actin cytoskeletal adaptors, ubiquitin ligases, and tyrosine kinases. The nature of these interactions suggests novel avenues to investigate the modulation of synaptic vesicle protein recycling.

EP News: Basic and Translational

Electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the Na channel gene SCN5A and its regulatory proteins have been linked to BrS. Aiba et al (Circ Cardiovasc Genet 2014;7:249, PMID 24795344) studied the properties of a BrS SCN5A mutation of a protein kinase A (PKA) consensus phosphorylation site, R526H. In vitro PKA phosphorylation was detected in the I-II linker of wild-type (WT) Na channels, but not in R526H or S528A mutants. Surface expression and peak Na current (INa) densities of R526H and S528A channels were reduced as compared with WT channels. Infection of rat cultures with WT and mutant channels increased conduction velocity compared with controls, but PKA stimulation significantly increased peak INa and conduction velocity only in WT cultures. With the intracellular application of NADH, both WT and mutant INa decrease, but this effect is reversed by PKA stimulation in WT channels but not in mutant channels. The authors conclude that the BrS mutation R526H is associated with a reduction in basal INa and a failure of PKA stimulation to augment the current, thus predisposing to arrhythmias in patients with BrS.

Pregabalin activates ROMK1 channels via cAMP-dependent protein kinase and protein kinase C

Pregabalin (PGB) displays analgesic and anticonvulsant activities. Regulation of the resting membrane potential (RMP) by renal outer medullary potassium (ROMK1) channels may provide a mechanism for these activities. We examined the effects of PGB on ROMK1 channel activity. To investigate the regulatory effect of PGB on the activity of ROMK1 channel, we used inside-out excised membrane patches to measure the K+ current in Xenopus oocytes expressed either the wild-type (WT) or mutant ROMK1 channels. PGB concentration-dependently enhanced the activity of ROMK1 channels. PGB increases the WT channels, pHi gating residue mutant channels (K80M) and the mutant channels at phosphatidylinositol bisphosphate (PIP2)-binding sites (R188Q, R217A, and K218A). Our study suggests that PGB in the regulating of ROMK1 channel function are neither by pHi- nor PIP2-dependent mechanism. We found PGB failed to prompt the activity of consensus phosphorylation sites for protein kinase C (PKC) mutated channels (S183A, T191A, T193A, S201A and T234A). Furthermore, PGB did not stimulate the activity of channels in the presence of cAMP-dependent protein kinase (PKA) inhibitors, the mutants of the C-terminal PKA-phosphorylation sites (S219A and S313A), and the mutants constructed (S219D and S313D) which mimic the addition of negative charged associated with phosphorylation bound to a serine. These results demonstrated that PKA- and PKC-mediated phosphorylation represents a novel mechanism for PGB-activated ROMK1 channels. The enhancement of ROMK1 currents proves to an important molecular mechanism underlying the analgesic/anticonvulsant property of PGB for the restoration of RMP.

Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) Increases the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Airway Epithelial Cells by Phosphorylating Shank2E Protein

The glucocorticoid dexamethasone increases cystic fibrosis transmembrane conductance regulator (CFTR) abundance in human airway epithelial cells by a mechanism that requires serum- and glucocorticoid-induced protein kinase 1 (SGK1) activity. The goal of this study was to determine whether SGK1 increases CFTR abundance by phosphorylating Shank2E, a PDZ domain protein that contains two SGK1 phosphorylation consensus sites. We found that SGK1 phosphorylates Shank2E as well as a peptide containing the first SGK1 consensus motif of Shank2E. The dexamethasone-induced increase in CFTR abundance was diminished by overexpression of a dominant-negative Shank2E in which the SGK1 phosphorylation sites had been mutated. siRNA-mediated reduction of Shank2E also reduced the dexamethasone-induced increase in CFTR abundance. Taken together, these data demonstrate that the glucocorticoid-induced increase in CFTR abundance requires phosphorylation of Shank2E at an SGK1 consensus site.

Peptide substrates for G protein-coupled receptor kinase 2.

G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the -2, -3, or -4 positions and its consensus phosphorylation site motifs were identified as (D/E)X1-3(S/T), (D/E)X1-3(S/T)(D/E), or (D/E)X0-2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (Km, 33.9 μM; Vmax, 0.35 pmol min(-1) mg(-1)), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.

A mutation causing Brugada syndrome identifies a mechanism for altered autonomic and oxidant regulation of cardiac sodium currents.

The mechanisms of the electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the sodium channel gene, SCN5A, and regulatory proteins that reduce or eliminate sodium current (INa) have been linked to BrS. We studied the properties of a BrS-associated SCN5A mutation in a protein kinase A (PKA) consensus phosphorylation site, R526H. In vitro PKA phosphorylation was detected in the I-II linker peptide of wild-type (WT) channels but not R526H or S528A (phosphorylation site) mutants. Cell surface expression of R526H and S528A channels was reduced compared with WT. Whole-cell INa through all channel variants revealed no significant differences in the steady-state activation, inactivation, and recovery from inactivation. Peak current densities of the mutants were significantly reduced compared with WT. Infection of 2D cultures of neonatal rat ventricular myocytes with WT and mutant channels increased conduction velocity compared with noninfected cells. PKA stimulation significantly increased peak INa and conduction velocity of WT but not mutant channels. Oxidant stress inhibits cardiac INa; WT and mutant INa decreases with the intracellular application of reduced nicotinamide adenine dinucleotide (NADH), an effect that is reversed by PKA stimulation in WT but not in R526H or S528A channels. We identified a family with BrS and an SCN5A mutation in a PKA consensus phosphorylation site. The BrS mutation R526H is associated with a reduction in the basal level of INa and a failure of PKA stimulation to augment the current that may contribute to the predisposition to arrhythmias in patients with BrS, independent of the precipitants.

S6 kinase 2 is bound to chromatin-nuclear matrix cellular fractions and is able to phosphorylate histone H3 at threonine 45 in vitro and in vivo.

The activity of S6 kinases (S6K) is highly induced in cancer cells highlighting an essential role in carcinogenesis. The S6K family has two members: S6K1 and S6K2 which bear common as well as distinct features. In an attempt to identify S6K2 unique sequence features compared to S6K1, we applied extensive bioinformatic analysis and motif search approaches. Interestingly, we identified 14 unique protein signatures which are present in proteins directly connected to chromatin and/or involved in transcription regulation. Using chromatin binding assay, we biochemically showed that S6K2 is bound to chromatin as well as nuclear matrix cellular fractions in HEK293 cells. The presence of S6K2 in chromatin fractions raised the possibility that it may be in close proximity to a number of chromatin substrates. For that, we then searched for S6K phosphorylation consensus sites RXRXXT/S in mammalian proteins using the SWISS-PROT database. Interestingly, we identified some potential phosphorylation sites in histone H3 (Thr45). Using in vitro kinase assays and siRNA-based knockdown strategy; we confirmed that S6K2 but not S6K1 or AKT is essential for histone H3-Thr45 phosphorylation in HEK293 cells. Furthermore, we show that the nuclear localisation sequence in the S6K2 C-terminus is essential for this modification. We have found that, H3-Thr45 phosphorylation correlates to S6K activation in response to mitogens and TPA-induced cell differentiation of leukaemic cell lines U937, HL60 and THP1. Overall, we demonstrate that S6K2 is a novel kinase that can phosphorylate histone H3 at position Thr45, which may play a role during cell proliferation and/or differentiation.

Phosphorylation of DEP-1/PTPRJ on threonine 1318 regulates Src activation and endothelial cell permeability induced by vascular endothelial growth factor

The protein tyrosine phosphatase DEP-1/PTPRJ positively regulates Src family kinases and critical biological functions in endothelial and hematopoietic cells. Phosphorylation of DEP-1 on Y1311/Y1320 mediates the association and activation of Src, and promotes Src-dependent angiogenic responses including endothelial cell permeability. We have identified T1318 as a phosphorylated residue proximal to Y1320. The aim of this study was to determine if T1318 phosphorylation exerts a regulatory role over the function of DEP-1. We show that phosphorylation of DEP-1 on Y1320 was reduced when T1318 was mutated. This led to the decreased association of DEP-1 T1318A with Src, and defective Src activation in both HEK 293T and VEGF-stimulated endothelial cells. Consistent with these findings, VEGF-induced tyrosine phosphorylation of VE-cadherin, its association to β-arrestin1/2, and cell permeability were impaired in cells expressing DEP-1 T1318A. Conversely, expression of the phosphomimetic mutant DEP-1 T1318E constitutively enhanced the phosphorylation of Y1320 and VE-cadherin over that induced by WT DEP-1, and resulted in increased VEGF-dependent permeability. DEP-1 T1318 is part of a CK2 consensus phosphorylation site and was identified as a CK2 substrate. Modulation of CK2 expression or activity in endothelial cells regulated T1318 phosphorylation, and correlated with the status of Y1320 phosphorylation, Src activation, and cell permeability. CK2-dependent phosphorylation of DEP-1 T1318 promotes Y1320 phosphorylation and Src activation upon VEGF stimulation. Phosphorylation of T1318 is thus part of a regulatory mechanism that channels the activity of DEP-1 towards Src to allow its optimal activation and the promotion of endothelial cell permeability.

The effects of artificial E-cadherin matrix-induced embryonic stem cell scattering on paxillin and RhoA activation via α-catenin

Mechanical forces have been shown to affect stem cell behavior in a large array of ways. However, our understanding of how these mechanical cues may regulate the behavior of embryonic stem cells (ESCs) remains in its infancy. Here, we aim to clarify the effect of cell scattering on the regulation of Rho family GTPases Rac1 and RhoA as well as paxillin. Allowing ESCs to spread and scatter on a synthetically designed E-cadherin substratum causes phosphorylation of paxillin on consensus phosphorylation sites leading to activation of Rac1 and inactivation of RhoA. By culturing cells in presence of RhoA activator or growing cells to a highly confluent state reverses the effect of cell scattering phenotype. Knockdown of E-cadherin-adapter protein α-catenin revealed that it negatively affects paxillin phosphorylation and up-regulates RhoA activity in compact cellular aggregates. Collectively these results indicate that cell scattering might cause a conformational change of α-catenin limiting its capacity to inhibit paxillin phosphorylation that causes an increase in Rac1 activation and RhoA deactivation. Understanding how synthetically designed extracellular matrix affect ESC signaling through mechanical cues brings a new aspect for stem cell engineers to develop technologies for controlling cell function.

Activation loop phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length

Specification of organelle size is crucial for cell function, yet we know little about the molecular mechanisms that report and regulate organelle growth and steady-state dimensions. The biflagellated green alga Chlamydomonas requires continuous-length feedback to integrate the multiple events that support flagellar assembly and disassembly and at the same time maintain the sensory and motility functions of the organelle. Although several length mutants have been characterized, the requisite molecular reporter of length has not been identified. Previously, we showed that depletion of Chlamydomonas aurora-like protein kinase CALK inhibited flagellar disassembly and that a gel-shift-associated phosphorylation of CALK marked half-length flagella during flagellar assembly. Here, we show that phosphorylation of CALK on T193, a consensus phosphorylation site on the activation loop required for kinase activity, is distinct from the gel-shift-associated phosphorylation and is triggered when flagellar shortening is induced, thereby implicating CALK protein kinase activity in the shortening arm of length control. Moreover, CALK phosphorylation on T193 is dynamically related to flagellar length. It is reduced in cells with short flagella, elevated in the long flagella mutant, lf4, and dynamically tracks length during both flagellar assembly and flagellar disassembly in WT, but not in lf4. Thus, phosphorylation of CALK in its activation loop is implicated in the disassembly arm of a length feedback mechanism and is a continuous and dynamic molecular marker of flagellar length during both assembly and disassembly.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

The Drosophila Nephrocyte

Drosophila nephrocytes have been an object of study for more than a century. They were named by Bruntz, based on observations of cells around the heart, the central nervous system, and in the sternal area in scorpions.[1][1] Kowalevsky suggested that they function as a storage kidney, because they

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

Requirement for protein kinase A in the phosphorylation of the TGFβ receptor-interacting protein km23-1 as a component of TGFβ downstream effects

km23-1 was previously identified as a TGFβ-receptor interacting protein that was phosphorylated on serines after TGFβ stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGFβ/protein kinase A (PKA) signaling. Using phosphorylation site prediction software, we found that km23-1 has two potential PKA consensus phosphorylation sites. In vitro kinase assays further demonstrated that PKA directly phosphorylates km23-1 on serine 73 (S73). Moreover, our results show that the PKA-specific inhibitor H89 diminishes phosphorylation of km23-1 on S73 after TGFβ stimulation. Taken together, our results demonstrate that TGFβ induction of PKA activity results in phosphorylation of km23-1 on S73. In order to assess the mechanisms underlying PKA phosphorylation of km23-1 on S73 (S73-km23-1) after TGFβ stimulation, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFβ regulates complex formation between the PKA regulatory subunit RIβ and km23-1 in vivo. In addition, an S73A mutant of km23-1 (S73A-km23-1), which could not be phosphorylated by PKA, inhibited TGFβ induction of the km23-1-dynein complex and transcriptional activation of the activin-responsive element (ARE). Furthermore, our results show that km23-1 is required for cAMP-responsive element (CRE) transcriptional activation by TGFβ, with S73-km23-1 being required for the CRE-dependent TGFβ stimulation of fibronectin (FN) transcription. Collectively, our results demonstrate for the first time that TGFβ/PKA phosphorylation of km23-1 on S73 is required for ARE- and CRE-mediated downstream events that include FN induction.

Double Mutation at the Putative Protein Kinase C Phosphorylation Sites Thr151 Plus Thr323 in the Mouse LeukotrieneD4 Receptor Eliminates Homologous Desensitization

Background/Aims: Signalling via CysLT1 is involved in activation of volume sensitive K⁺ channels and homologous desensitization of the LTD₄ receptor impairs regulatory volume decrease (RVD). The aim is to illustrate the effect of mutation of putative PKC consensus phosphorylation sites in the CysLT1R on desensitization and RVD. Methods: mCysLT1 contains 4 putative PKC consensus phosphorylation sites, and four mutants were created: Thr151Gly, Thr323Gly, Thr151Gly plus Thr323Gly, and Thr236Gly plus Ser243Gly. Functional mCysLT1 receptor activity after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was visualized as a LTD₄-evoked, Ca²⁺-activated Cl^- currents recorded by two-electrode voltage clamp. Results: Repetitive LTD₄ administration (100 nM) desensitized the LTD₄-evoked currents in oocytes expressing wild type CysLT1. Single mutations as well as the double mutation Thr236Gly plus Ser243Gly had no or a slight effect on the LTD₄ induced desensitization. However, double mutation Thr323Gly plus Thr151Gly prevented the desensitization. As a functional consequence we find that inhibition of PKC accelerates RVD and prevents the inhibitory effect of LTD₄-pretreatment on RVD in Ehrlich ascites tumour cells. Conclusion: These data indicate that simultaneous PKC-mediated phosphorylation at the 2^nd inner loop (Thr¹⁵¹) and at the C-terminal domain (Thr³²³) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling.

Lipid-dependent and -independent regulation of nuclear envelope disassembly

The nuclear envelope(NE) isanorganelle ofuniquecomplexand dynamic structure, which serves not only as a physicalbarrier between the nucleus and the cytoplasm, but also as aregulator of many cellular events. During open mitosis, theNE is disassembled in prophase in order to allow the mitoticspindletoaccessthechromosomes[1].NEbreakdownentailsthe disassembly of all the NE components and is associatedwith a series of phosphorylations of proteins of the innernuclear membrane [2], nuclear pore complex [3]andlamina[4], the latest promoting depolymerisation of the lamin fila-ments. The A-type lamins are then solubilised in the cyto-plasm, whereas B-type lamins disperse in the endoplasmicreticulum membranes [5]. Multiple kinases, including cyclin-dependent kinase1(CDK1)anddiacylglycerol-activatedpro-tein kinase Cs (PKCs), have been suggested to play a role inlaminphosphorylationanddisassemblyinvariousmodelsandorganisms,mostlyin cell-free systems [6–17].InCaenorhab-ditiselegans,ahomologueoflipin,alipidphosphatasewhichconverts phosphatidic acid to DAG, is required for lamindisassembly during NE breakdown [18, 19]. To investigatemitotic lamin B1 disassembly, Mall et al. have developed ahigh-throughputlive-imagingassay[20].ThedataobtainedinHeLa cells using this assay confirm previous results suggest-ing the involvement of CDK1, PKCs and lipins in lamindisassembly.This assay is based on the automatic tracking and classi-fication intomitotic phases ofhundredsof individual mitoticcells expressing the chromatin marker H2B and lamin B1chimaeras, monitored by wide-field microscopy. Briefly,nuclei were detected in the H2B-mCherry channel,classified as previously described [21] and the chromatinwas segmented. A ring around the chromatin was defined tomeasure GFP-lamin B1 fluorescence intensity and deducethe assembled or disassembled (in the cytoplasm) state oflamin B1 throughout mitosis. Using a mathematical model,the authors determined the durations of lamin disassemblyin each condition. Mitotic progression from prophase toanaphase and chromosome congression (mean duration ofprometaphase and metaphase) were also calculated.Results obtained with transiently transfected wild-type ormutant lamin B1 reporter using this assay or higher resolu-tion images acquired by confocal microscopy showed thatmutation of the PKC consensus phosphorylation site did notalter lamin disassembly, whereas mutation of the CDK1phosphorylation site alone or combined to the mutation ofthe PKC motif significantly delayed lamin B1 disassemblycompared to controls. However, the differences in the meanduration of lamin disassembly between wild-type andCDK1- or CDK1/PKC-phosphorylation site mutants weresubtle, although significant (7.51±0.87 and 8.24±0.79 minp00.0146 or 8.86±0.80 min, p<0.0001, respectively). Theweakness of the mutants might be attributed to insufficientlevels of expression compared to endogenous proteins(Western blots omitted) and inability to form a network ofmutant proteins which would resist lamina disassembly.Another possibility is that these phosphorylation sites, onlyplay a minor role in lamin disassembly. Relatively highdoses (compared to IC50) of kinase inhibitors, i.e. classicalPKC inhibitor Go6976 and CDK1 inhibitor roscovitinealone or combined,caused significantperturbations of lamindisassembly and mitotic progression, although the twoevents were not correlated, and induced the apparition of‘cytoplasmic fragments’ of lamin B1. Those high intensity‘cytoplasmic fragments’, which were also observed by con-focal microscopy with the CDK1- or CDK1/PKC-sitemutants, could correspond to aggregates of lamin B1 in