Consensus Phosphorylation Sites Research Articles

SGK1 Kinase Upregulates GLUT1 Activity and Plasma Membrane Expression

Phosphatidylinositol 3-kinase (PI3 kinase) inhibition disrupts the ability of insulin to stimulate GLUT1 and GLUT4 translocation into the cell membrane and thus glucose transport. The effect on GLUT4 but not on GLUT1 is mediated by activation of protein kinase B (PKB). The serum- and glucocorticoid-inducible kinase SGK1, a further kinase downstream of PI3 kinase, regulates several transporters by enhancing their plasma membrane abundance. GLUT1 contains a consensus site ((95)Ser) for phosphorylation by SGK1. Thus, the present study investigated whether GLUT1 is regulated by the kinase. Tracer-flux studies in Xenopus oocytes and HEK-293 cells demonstrated that GLUT1 transport is enhanced by constitutively active (S422D)SGK1. The effect requires the kinase catalytical activity since the inactive mutant (K127N)SGK1 failed to modulate GLUT1. GLUT1 stimulation by (S422D)SGK1 is not due to de novo protein synthesis but rather to an increase of the transporter's abundance in the plasma membrane. Kinetic analysis revealed that SGK1 enhances maximal transport rate without altering GLUT1 substrate affinity. These observations suggest that SGK1 regulates GLUT1 and may contribute to or account for the PI3 kinase-dependent but PKB-independent stimulation of GLUT1 by insulin.

Extrasynaptic Membrane Trafficking Regulated by GluR1 Serine 845 Phosphorylation Primes AMPA Receptors for Long-term Potentiation

Enhancement of synaptic transmission, as occurs in long-term potentiation (LTP), can result from several mechanisms that are regulated by phosphorylation of the AMPA-type glutamate receptor (AMPAR). Using a quantitative assay of net serine 845 (Ser-845) phosphorylation in the GluR1 subunit of AMPARs, we investigated the relationship between phospho-Ser-845, GluR1 surface expression, and synaptic strength in hippocampal neurons. About 15% of surface AMPARs in cultured neurons were phosphorylated at Ser-845 basally, whereas chemical potentiation (forskolin/rolipram treatment) persistently increased this to 60% and chemical depression (N-methyl-D-aspartate treatment) decreased it to 10%. These changes in Ser-845 phosphorylation were paralleled by corresponding changes in the surface expression of AMPARs in both cultured neurons and hippocampal slices. For every 1% increase in net phospho-Ser-845, there was 0.75% increase in the surface fraction of GluR1. Phosphorylation of Ser-845 correlated with a selective delivery of AMPARs to extrasynaptic sites, and their synaptic localization required coincident synaptic activity. Furthermore, increasing the extrasynaptic pool of AMPA receptors resulted in stronger theta burst LTP. Our results support a two-step model for delivery of GluR1-containing AMPARs to synapses during activity-dependent LTP, where Ser-845 phosphorylation can traffic AMPARs to extrasynaptic sites for subsequent delivery to synapses during LTP.

First joint Italian–German purine club meeting “Progress in Purinergic Receptor Pharmacology and Function”

First joint Italian–German purine club meeting “Progress in Purinergic Receptor Pharmacology and Function”

Conformational Changes of Yeast Plasma Membrane H+-ATPase during Activation by Glucose: Role of Threonine-912 in the Carboxy-Terminal Tail

Yeast Pma1 H(+)-ATPase, which belongs to the P-type family of cation-transporting ATPases, is activated up to 10-fold by growth on glucose, and indirect evidence has linked the activation to Ser/Thr phosphorylation within the C-terminal tail. We have now used limited trypsinolysis to map glucose-induced conformational changes throughout the 100 kDa ATPase. In the wild-type enzyme, trypsin cleaves first at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavages occur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6-M7 cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At the C-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes from glucose-starved cells (GS) but becomes accessible in membranes from glucose-metabolizing cells (GM). In the presence of orthovanadate, Lys-174 at the boundary between M2 and the A domain also becomes open to cleavage in GM but not GS samples (site T5). Significantly, this global conformational change can be suppressed by mutations at Thr-912, a consensus phosphorylation site near the C-terminus. Substitution by Ala at position 912 leads to a GS-like (trypsin-resistant) state, while substitution by Asp leads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecular movements that result in glucose activation.

Cross-talk between the Two Divergent Insulin Signaling Pathways Is Revealed by the Protein Kinase B (Akt)-mediated Phosphorylation of Adapter Protein APS on Serine 588

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

Cdc28-Dependent Regulation of the Cdc5/Polo Kinase

Polo kinase is activated as cells enter mitosis and plays a central role in coordinating diverse mitotic events, yet the mechanisms leading to activation of Polo kinase are poorly understood . Work in Xenopus meiotic cell cycles has suggested that Polo kinase functions in a pathway that helps trigger activation of Cdk1 . However, studies in other organisms have suggested that activation of Polo kinase is dependent upon Cdk1 and therefore occurs downstream of Cdk1 activation . In this study, we have investigated the role of Cdk1 in the activation of budding yeast Polo kinase. The budding yeast homologs of Cdk1 and Polo kinase are referred to as Cdc28 and Cdc5. We show that signaling from Cdc28 is required to maintain Cdc5 activity in vivo. Furthermore, purified Cdc28 associated with the mitotic cyclin Clb2 is sufficient to activate purified Cdc5 in vitro. A single Cdc28 consensus phosphorylation site found at threonine 242 in the activation loop segment of Cdc5 is required for Cdc5 function in vivo and for kinase activity in vitro, whereas four other Cdc28 consensus sites are dispensable. Analysis of Cdc5 phosphorylation by mass spectrometry indicates that threonine 242 is phosphorylated in vivo. These results suggest that Cdc28 activates Cdc5 via phosphorylation of threonine 242.

Decrease of current responses at human recombinant P2X 3 receptors after substitution by Asp of Ser/Thr residues in protein kinase C phosphorylation sites of their ecto-domains

The whole-cell patch-clamp technique was used to record current responses to nucleotides in HEK 293 cells transiently transfected with the human (h) P2X 3 receptor. When GDP-β-S was included into the pipette solution, UTP at concentrations which did not alter the holding current, facilitated the α,β-methylene ATP (α,β-meATP)-induced current. The substitution of Ser/Thr residues situated within protein kinase C (PKC) consensus phosphorylation sites of the P2X 3 receptor ecto-domain by the neutral amino acid Ala either abolished (T134A, S178A) or did not alter (T196A, S269A) the UTP-induced potentiation of the α,β-meATP current. The substitution of the same Ser/Thr residues in all four PKC sites by the negatively charged Asp prevented the potentiation by UTP. The Asp mutations abolished the first, fast offset time-constant, but did not alter, or in the case of S269D even increased, the second, slow offset time-constant; at the same time such mutations invariably increased the onset time-constant and massively depressed the peak current amplitude. None of the Ala mutations (with the exception of S269A) influenced the time-course of desensitisation or the peak current amplitude. It is concluded that constitutive activation of PKC sites at the ecto-domain of the hP2X 3 receptor both abolishes the UTP-induced potentiation of the α,β-meATP current and accelerates its rate of desensitisation.

Regulation of Yeast mRNA 3′ End Processing by Phosphorylation

Recent studies have found that the phosphatase Glc7 associates with the yeast cleavage/polyadenylation factor (CPF), but the role of Glc7 in 3' end processing has not been investigated. Here, we report that depletion of Glc7 causes shortened poly(A) tails in vivo and accumulation of phosphorylated Pta1, a CPF subunit. Removal of Glc7 also gives extract defective for poly(A) addition but normal for cleavage at the poly(A) site. Polyadenylation is rescued by addition of Glc7 or Pta1, but not by phosphorylated Pta1. Moreover, Ypi1, a Glc7-specific inhibitor, or the Cka1 kinase blocks poly(A) addition in wild-type (wt) extract. Pta1 interacts physically and genetically with Glc7, suggesting that Pta1 may also regulate Glc7 or recruit it to CPF. A weakened association of Fip1 with phosphorylated CPF may explain the specific effect on polyadenylation. These results support a model in which poly(A) synthesis is controlled by cycles of phosphorylation and dephosphorylation that require the action of Glc7.

Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an α-Helical Conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.

A Role for Proapoptotic BID in the DNA-Damage Response

The BCL-2 family of apoptotic proteins encompasses key regulators proximal to irreversible cell damage. The BH3-only members of this family act as sentinels, interconnecting specific death signals to the core apoptotic pathway. Our previous data demonstrated a role for BH3-only BID in maintaining myeloid homeostasis and suppressing leukemogenesis. In the absence of Bid, mice accumulate chromosomal aberrations and develop a fatal myeloproliferative disorder resembling chronic myelomonocytic leukemia. Here, we describe a role for BID in preserving genomic integrity that places BID at an early point in the path to determine the fate of a cell. We show that BID plays an unexpected role in the intra-S phase checkpoint downstream of DNA damage distinct from its proapoptotic function. We further demonstrate that this role is mediated through BID phosphorylation by the DNA-damage kinase ATM. These results establish a link between proapoptotic Bid and the DNA-damage response.

Protein Kinase A-dependent Phosphorylation of Lutheran/Basal Cell Adhesion Molecule Glycoprotein Regulates Cell Adhesion to Laminin α5

Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha5 in sickle cell disease.

Interactions Between Kinases and Phosphatases in the Rapid Control of Brain Aromatase

Aromatization of testosterone into oestradiol plays a key role in the activation of male sexual behaviour in many vertebrate species. Rapid changes in brain aromatase activity have recently been identified and the resulting changes in local oestrogen bioavailability could modulate fast behavioural responses to oestrogens. In quail hypothalamic homogenates, aromatase activity is down-regulated within minutes by calcium-dependent phosphorylations in the presence of ATP, MgCl2 and CaCl2 (ATP/Mg/Ca). Three kinases (protein kinases A and C and calmodulin kinase; PKA, PKC and CAMK) are potentially implicated in this process. If kinases decrease aromatase activity in a reversible manner, then it would be expected that the enzymatic activity would increase and/or return to baseline levels in the presence of phosphatases. We showed previously that 0.1 mM vanadate (a general inhibitor of protein phosphatases) significantly decreases aromatase activity but specific protein phosphatases that could up-regulate aromatase activity have not been identified to date. The reversibility of aromatase activity inhibition by phosphorylations was investigated in the present study using alkaline and acid phosphatase (Alk and Ac PPase). Unexpectedly, Alk PPase inhibited aromatase activity in a dose-dependent manner in the presence, as well as in the absence, of ATP/Mg/Ca. By contrast, Ac PPase completely blocked the inhibitory effects of ATP/Mg/Ca on aromatase activity, even if it moderately inhibited aromatase activity in the absence of ATP/Mg/Ca. However, the addition of Ac PPase was unable to restore aromatase activity after it had been inhibited by exposure to ATP/Mg/Ca. Taken together, these data suggest that, amongst the 15 potential consensus phosphorylation sites identified on the quail aromatase sequence, some must be constitutively phosphorylated for the enzyme to be active whereas phosphorylation of the others is involved in the rapid inhibition of aromatase activity by the competitive effects of protein kinases and phosphatases. Two out of these 15 putative phosphorylation sites occur in an environment corresponding to the consensus sites for PKC, PKA (and possibly a CAMK) and, in all probability, represent the sites whose phosphorylation rapidly blocks enzyme activity.

Functional characterization of Schizosaccharomyces pombe neutral trehalase altered in phosphorylatable serine residues

The activation of neutral trehalase (Ntp1) by metabolic and physical stresses in Schizosaccharomyces pombe is dependent on protein kinases Pka1 or Sck1. Mutant ntp1 alleles altered for potentially phosphorylatable serine residues within the regulatory domain of the enzyme were integrated under the control of the native promoter in an ntp1-deleted background. The trehalase variants were expressed to a level similar to that of wild type trehalase from control cells. Wild type trehalase protein accumulated and became activated upon stress while a single change in the evolutionary conserved perfect consensus site for Pka1-dependent phosphorylation (Ser71), as well as point mutations in two other putative phosphorylation sites (Ser6, Ser51), produced inactive trehalases unresponsive to stress. Trehalose content in the trehalase mutated strains increased upon salt stress to a level comparable to that shown by an ntp1-deleted mutant. When exposed to heat shock, trehalose hyperaccumulated in the ntp1-null strain lacking trehalase protein and this phenotype was shown by some (Ser71), but not all, strains with serine mutated trehalases. The mutant trehalases retained the ability to form complexes with trehalose-6-phosphate synthase. These data support a role of potentially phosphorylated specific sites for the activation of S. pombe neutral trehalase and for the heat shock-induced accumulation of trehalose.

ArgBP2γ Interacts with Akt and p21-activated Kinase-1 and Promotes Cell Survival

Akt/protein kinase B is a major cell survival pathway through phosphorylation of proapoptotic proteins Bad and Bax and of additional apoptotic pathways linked to Forkhead proteins glycogen synthase kinase-3beta and ASK1. To further explore the mechanism by which Akt regulates cell survival, we identified an Akt interaction protein by yeast two-hybrid screening. It is highly homologous to ARG-binding protein 2 (ArgBP2) with splicing exon 8 of the coding region of the ArgBP2. As two splicing isoforms (ArgBP2alpha and -beta) of ArgBP2 have been identified (Wang, B., Golemis, E. A., and Kruh, G. D. (1997) J. Biol. Chem. 272, 17542-17550), it was named ArgBP2gamma. ArgBP2gamma contains four Akt phosphorylation consensus sites, a SoHo motif, and three Src homology (SH) 3 domains and binds to C-terminal proline-rich motifs of Akt through its first and second SH3 domains. It also interacts with p21-activated protein kinase (PAK1) via its first and third SH3 domains, indicating the SH3 domains of ArgBP2gamma as docking sites for Akt and PAK1. Akt phosphorylates ArgBP2gamma in vitro and in vivo. Expression of ArgBP2gamma induces PAK1 activity and overrides apoptosis induced by ectopic expression of Bad or DNA damage. Nonphosphorylatable ArgBP2gamma-4A and SH3 domain-truncated mutant ArgBP2gamma inhibit Akt-induced PAK1 activation and reduce Akt and PAK1 phosphorylation of Bad and antiapoptotic function. These data indicate that ArgBP2gamma is a physiological substrate of Akt, functions as an adaptor for Akt and PAK1, and plays a role in Akt/PAK1 cell survival pathway.

Polo-like Kinase 1 Creates the Tension-Sensing 3F3/2 Phosphoepitope and Modulates the Association of Spindle-Checkpoint Proteins at Kinetochores

In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression. We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest. Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.

Constitutively Active Homo-oligomeric Angiotensin II Type 2 Receptor Induces Cell Signaling Independent of Receptor Conformation and Ligand Stimulation

Members of the G-protein-coupled receptor superfamily (GPCRs) undergo homo- and/or hetero-oligomerization to induce cell signaling. Although some of these show constitutive activation, it is not clear how such GPCRs undergo homo-oligomerization with transmembrane helix movement. We previously reported that angiotensin II (Ang II) type 2 (AT(2)) receptor, a GPCR, showed constitutive activation and induced apoptosis independent of its ligand, Ang II. In the present study, we analyzed the translocation and oligomerization of the AT(2) receptor with transmembrane movement when the receptor induces cell signaling. Constitutively active homo-oligomerization, which was due to disulfide bonding between Cys(35) in one AT(2) receptor and Cys(290) in another AT(2) receptor, was localized in the cell membrane without Ang II stimulation and induced apoptosis without changes in receptor conformation. These results provide the direct evidence that the constitutively active homo-oligomeric GPCRs by intermolecular interaction in two extracellular loops is translocated to the cell membrane and induces cell signaling independent of receptor conformation and ligand stimulation.

Light-independent Phosphorylation of WHITE COLLAR-1 Regulates Its Function in the Neurospora Circadian Negative Feedback Loop

Phosphorylation is a major regulatory mechanism controlling circadian clocks. In the Neurospora circadian clock, the PER-ARNT-SIM (PAS) domain-containing transcription factor, WHITE COLLAR (WC)-1, acts both as the blue light photoreceptor of the clock and as a positive element in the circadian negative feedback loop in constant darkness, by activating the transcription of the frequency (frq) gene. To understand the role of WC-1 phosphorylation, five in vivo WC-1 phosphorylation sites, located immediately downstream of the WC-1 zinc finger DNA binding domain, were identified by tandem mass spectrometry using biochemically purified endogenous WC-1 protein. Mutations of these phosphorylation sites suggest that they are major WC-1 phosphorylation sites under constant conditions but are not responsible for the light-induced hyperphosphorylation of WC-1. Although phosphorylation of these sites does not affect the light function of WC-1, strains carrying mutations of these sites show short period, low amplitude, or arrhythmic conidiation rhythms in constant darkness. Furthermore, normal or slightly higher levels of frq mRNA and FRQ proteins were observed in a mutant strain containing mutations of all five sites despite its low WC-1 levels. Together, these data suggest that phosphorylation of these sites negatively regulates the function of WC-1 in the circadian negative feedback loop and is important for the function of the Neurospora circadian clock.

Drebrin E2 is differentially expressed and phosphorylated in parietal cells in the gastric mucosa

Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.

E47 phosphorylation by p38 MAPK promotes MyoD/E47 association and muscle-specific gene transcription

Selective recognition of the E-box sequences on muscle gene promoters by heterodimers of myogenic basic helix-loop-helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are unable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. Here we show that p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle-specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro-myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis.

In vivo analysis of the roles of conserved aspartate and histidine residues within a complex response regulator

RcaC is the founding member of a group of large response regulators with complex domain combinations containing at least two receiver domains, an OmpR-class winged helix-turn-helix DNA binding domain, and a histidine phosphotransfer (HPt) domain. Within its two receiver and HPt domains, RcaC contains consensus phosphorylation sites at aspartates 51, 576 and histidine 316. RcaC operates in the pathway regulating transcription of genes encoding components of photosynthetic light harvesting antenna to changes in light colour. We show that phycocyanin gene expression requires RcaC. RcaC contributes to light regulation of phycoerythrin genes, but is not part of the second light regulation pathway controlling these genes. Substitutions at aspartate 51 or histidine 316 severely impaired light responsiveness while substitutions at aspartate 576 had little effect. Complete loss of light regulation, measured by phycocyanin gene expression, only occurred in the triple mutant. We conclude that aspartate 51 primarily controls light colour responsiveness and is regulated by histidine 316, and that these residues are likely phosphorylated in red light and dephosphorylated in green light. The carboxy-terminal receiver domain has a minor role in controlling this response. RcaC abundance is also light regulated and depends on aspartate 51 and histidine 316, but not aspartate 576.