Biomedical subjects

H Mellor

Publications and source records attributed to H Mellor.

At least 19 recordsLinked to original sources

PubMed2006

Expression of RND proteins in human myometrium.

RHO GTPases are key regulators of the actin cytoskeleton and stress fiber formation. In the human uterus, activated RHOA forms a complex with RHO-associated protein kinase (ROCK) which inhibits myosin light chain phosphatase (PPP1R12A), causing a calcium-independent increase in myosin light chain phosphorylation and tension (Ca2+ sensitization). Recently discovered small GTP binding RND proteins can inhibit RHOA and ROCK interaction to reduce calcium sensitization. Very little is known about the expression of RND proteins in the human uterus. We tested the hypothesis that the uterine quiescence observed during gestation is mediated by an increase in RND protein expression inhibiting RHOA-ROCK-mediated PPP1R12A phosphorylation. Immunohistochemistry and immunoblotting were used to determine RHOA and RND protein expression and localization in nonpregnant, pregnant nonlaboring, and laboring patients at term and patients in spontaneous preterm labor. Changes in protein expression estimated by densitometry between different patient groups were measured. A significant increase of RND2 and RND3 protein expression was observed in pregnant relative to nonpregnant myometrium associated with a loss of PPP1R12A phosphorylation. RND transfected myometrial cells demonstrated a dramatic loss of stress fiber formation and a "rounding" phenotype. RND upregulation in pregnancy may inhibit RHOA-ROCK-mediated increase in calcium sensitization to facilitate the uterine quiescence observed during gestation.

Adult↗

PubMed2004

What is in a filopodium? Starfish versus hedgehogs.

Many cell types can generate thin actin-based protrusive structures, which are often classified under the general term of 'filopodia'. However, a range of filopodia-like structures exists that differ both morphologically and functionally. In this brief review, we discuss the different types of filopodial structures, together with the actin-binding proteins and signalling pathways involved in their formation. Specifically, we highlight the differences between the filopodial extensions induced by the Rho GTPases Cdc42 and Rif.

Actins↗

PubMed2002

Functional coupling of rat metabotropic glutamate 1a receptors to phospholipase D in CHO cells: involvement of extracellular Ca2+, protein kinase C, tyrosine kinase and Rho-A.

We report here that metabotropic glutamate 1a (mGlu1a) receptors, stably expressed in CHO cells, stimulate phospholipase D (PLD) activity. Several mGlu receptor agonists were found to exert this effect, with a rank order of potency of: L-quisqualate>L-glutamate>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]=(S)-3,5-dihydroxyphenylglycine [(S)-DHPG]. Both L-glutamate- and (1S,3R)-ACPD-stimulated PLD activity were attenuated by the selective mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine. mGlu1a receptor-stimulated PLD was inhibited either by the selective protein kinase C (PKC) inhibitor, GF109203X, or via PKC downregulation. MGlu1a receptor-PLD coupling required extracellular Ca2+ and was sensitive to La3+ and Zn2+, inhibitors of intracellular Ca2+ store-operated Ca2+ influx. mGlu1a receptor-PLD coupling was inhibited by the selective tyrosine kinase inhibitor, genistein. In addition, mGlu1a receptor-PLD coupling was also inhibited by cell transfection with the selective Rho (small GTP-binding protein) inhibitors: C3-exoenzyme and dominant negative mutant RhoA constructs. Brefeldin A, a selective ADP-ribosylation factor (ARF) inhibitor, and a dominant negative ARF6 mutant, failed to significantly influence mGlu1a receptor-stimulated PLD activity. We conclude that mGlu1a receptors activate PLD via a mechanism that is dependent on extracellular Ca2+, PKC, tyrosine kinase and RhoA but independent of ARF.

Animals↗

PubMed2001

Modular phosphoinositide-binding domains--their role in signalling and membrane trafficking.

The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.

Amino Acid Sequence↗

PubMed2001

Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1).

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements.

Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.

Actins↗

PubMed2000

Rho GTPase control of protein kinase C-related protein kinase activation by 3-phosphoinositide-dependent protein kinase.

The protein kinase C-related protein kinases (PRKs) have been shown to be under the control of the Rho GTPases and influenced by autophosphorylation. In analyzing the relationship between these inputs, it is shown that activation in vitro and in vivo involves the activation loop phosphorylation of PRK1/2 by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Rho overexpression in cultured cells is shown to increase the activation loop phosphorylation of endogenous PRKs and is demonstrated to influence this process by controlling the ability of PRKs to bind to PDK1. The interaction of PRK1/2 with PDK1 is shown to be dependent upon Rho. Direct demonstration of ternary (Rho.PRK.PDK1) complex formation in situ is provided by the observation that PDK1 is recruited to RhoB-containing endosomes only if PRK is coexpressed. Furthermore, this in vivo complex is maintained after phosphoinositide 3-kinase inhibition. The control of PRKs by PDK1 thus evidences a novel strategy of substrate-directed control involving GTPases.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2000

Regulation of endocytic traffic by rho family GTPases.

Endocytosis is a complicated yet highly efficient process that involves the uptake and processing of cargoes, ranging from small molecules, to activated signalling receptors, to whole microorganisms. Regulation of endocytic pathways is poorly understood. Recent evidence suggests that the Rho GTPase family of signalling proteins is intimately involved in endocytic traffic, providing novel insights into the control mechanisms that govern this process.

3T3 Cells↗

PubMed2000

Rho-dependence of Schizosaccharomyces pombe Pck2.

BACKGROUND: In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C-related PRKs, is responsible for an interaction with Rho-GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho-dependent protein kinase C-related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2. RESULTS: Rho1 is shown to interact with the conserved N-terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co-expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2-Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located. CONCLUSIONS: The results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho-GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.

Amino Acid Sequence↗

PubMed1999

Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres. It is also involved in other cell signalling pathways including the regulation of gene expression and the generation of lipid second messengers [1] [2]. RhoA is very closely related to two other small GTPases about which much less is known: RhoB and RhoC (which are approximately 83% identical). Perhaps the most intriguing of these is RhoB. RhoA is largely cytosolic but translocates to the plasma membrane on activation. RhoB, however, is entirely localised to the cytosolic face of endocytic vesicles [3] [4]. This suggests a potential role for RhoB in regulating endocytic traffic; however, no evidence has been presented to support this. RhoA has been shown to act at the plasma membrane to regulate the clathrin-mediated internalisation of transferrin receptor [5] and of the muscarinic acetylcholine receptor [6]. We have recently demonstrated that RhoB binds the RhoA effector, PRK1 and targets it to the endosomal compartment [7]. We show here that RhoB acts through PRK1 to regulate the kinetics of epidermal growth factor receptor traffic.

Amides↗

PubMed1999

Imaging protein kinase Calpha activation in cells.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.

3T3 Cells↗

PubMed1998

The extended protein kinase C superfamily.

Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in a multitude of cellular processes, ranging from control of fundamental cell autonomous activities, such as proliferation, to more organismal functions, such as memory. However, understanding of mammalian PKC signalling systems is complicated by the large number of family members. Significant progress has been made through studies based on comparative analysis, which have defined a number of regulatory elements in PKCs which confer specific location and activation signals to each isotype. Further studies on simple organisms have shown that PKC signalling paradigms are conserved through evolution from yeast to humans, underscoring the importance of this family in cellular signalling and giving novel insights into PKC function in complex mammalian systems.

Amino Acid Sequence↗

PubMed1998

PRK1 is targeted to endosomes by the small GTPase, RhoB.

RhoB has been shown to be an endosomal GTPase both by immunocytochemistry and electron microscopy, however, its role in endocytosis is unknown. Elucidation of the cellular roles of other members of this superfamily of signaling proteins has come with the identification of their downstream partners. We show here that the recently isolated serine/threonine kinase PRK1 is targeted to the endosomal compartment by RhoB. This is established both through immunofluorescence and cell fractionation. PRK1 is shown to interact with activated RhoB in cells and is localized to endosomes through its Rho-binding HR1 domain. Translocation of PRK1 to the endosomal compartment by RhoB is accompanied by a shift in the electrophoretic mobility of the kinase indicative of an accompanying activation.

3T3 Cells↗

PubMed1998

Multiple interactions of PRK1 with RhoA. Functional assignment of the Hr1 repeat motif.

PRK1 (PKN) is a serine/threonine kinase that has been shown to be activated by RhoA (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650). Detailed analysis of the PRK1 region involved in RhoA binding has revealed that two homologous sequences within the HR1 domain (HR1a and HR1b) both bind to RhoA; the third repeat within this domain, HR1cPRK1, does not bind RhoA. The related HR1 motif is also found to confer RhoA binding activity to the only other fully cloned member of this kinase family, PRK2. Furthermore, the predictive value of this motif is established for an HR1a sequence derived from a Caenorhabditis elegans open reading frame encoding a protein kinase of unknown function. Interestingly, the HR1aPRK1 and HR1bPRK1 subdomains are shown to display a distinctive nucleotide dependence for RhoA binding. HRIaPRK1 is entirely GTP-dependent, while HR1bPRK1 binds both GTP- and GDP-bound forms of RhoA. This distinction indicates that there are two sites of contact between RhoA and PRK1, one contact through a region that is conformationally dependent upon the nucleotide-bound state of RhoA and one that is not. Analysis of binding to Rho/Rac chimera provides evidence for a HR1aPRK1 but not HR1bPRK1 interaction in the central third of Rho. Additionally, it is observed that the V14RhoA mutant binds HR1a but does not bind HR1b. This distinct binding behavior corroborates the conclusion that there are independent contacts on RhoA for the HR1aPRK1 and HR1bPRK1 motifs.

Amino Acid Sequence↗

PubMed1997

Specific proteolysis of the kinase protein kinase C-related kinase 2 by caspase-3 during apoptosis. Identification by a novel, small pool expression cloning strategy.

The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translated in vitro in the presence of [35S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp117 and Asp700 in vitro. In addition, PRK2 is cleaved rapidly during Fas- and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.

Amino Acid Chloromethyl Ketones↗

PubMed1996

eIF2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2. Sequence conservation between the alpha, beta and delta subunits of eIF2B from mammals and yeast.

The guanine nucleotide-exchange factor eIF2B mediates the exchange of GDP bound to translation initiation factor eIF2 for GTP. This exchange process is a key regulatory step for the control of translation initiation in eukaryotic organisms. To improve our understanding of the structure, function and regulation of eIF2B, we have obtained and sequenced cDNA species encoding all of its five subunits. Here we report the sequences of eIF2B beta and delta from rat. This paper focuses on sequence similarities between the alpha, beta and delta subunits of mammalian eIF2B. Earlier work showed that the amino acid sequences of the corresponding subunits of eIF2B in the yeast Saccharomyces cerevisiae (GCN3, GCD7 and GCD2) exhibit considerable similarity. We demonstrate that this is also true for the mammalian subunits. Moreover, alignment of the eIF2B alpha, beta and delta sequences from mammals and yeast, along with the sequence of the putative eIF2B alpha subunit from Caenorhabditis elegans and eIF2B delta from Schizosaccharomyces pombe shows that a large number of residues are identical or conserved between the C-terminal regions of all these sequences. This strong sequence conservation points to the likely functional importance of these residues. The implications of this are discussed in the light of results concerning the functions of the subunits of eIF2B in yeast and mammals. Our results also indicate that the large apparent differences in mobility on SDS/PAGE between eIF2B beta and delta subunits from rat and rabbit are not due to differences in their lengths but reflect differences in amino acid composition. We have also examined the relative expression of mRNA species encoding the alpha, beta, delta and epsilon subunits of eIF2B in a range of rat tissues by Northern blot analysis. As might be expected for mRNA species encoding subunits of a heterotrimeric protein, the ratios of expression levels of these subunits to one another did not vary between the different rat tissues examined (with the possible exception of liver). This represents the first analysis of the levels of expression of mRNA species encoding the different subunits of eIF2B.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of cDNAs encoding the epsilon-subunit of eukaryotic initiation factor-2B from rabbit and human.

A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of complementary and genomic DNAs encoding the epsilon-subunit of rat translation initiation factor-2B.

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide-exchange protein involved in the recycling of eIF-2 during peptide-chain initiation. Regulation of eIF-2B activity occurs under a wide range of conditions by diverse mechanisms. To better understand the regulation of eIF-2B activity as well as the coordinate expression of its five subunits, we have begun to clone and characterize the cDNAs and genes encoding these proteins. In the present study, complementary and genomic DNAs encoding the epsilon-subunit of rat eIF-2B were cloned and characterized. The cDNA is 2517 bp in length, including a 30 nt poly(A) tail, and recognizes both 2.7 and 3.5 kb mRNA species on Northern blots of rat RNA. The cDNA contains a 2151 bp open reading frame encoding 716 amino acids producing a protein with a predicted molecular mass of 80 kDa. The derived amino acid sequence contains regions identical to three peptides obtained from bovine liver eIF-2B epsilon and is 31% identical to Gcd6, the putative yeast eIF-2B epsilon. Examination of the derived amino acid sequence of rat eIF-2B epsilon reveals phosphorylation site motifs for several protein kinases which have been implicated in regulation of guanine nucleotide exchange activity. The mRNA for eIF-2B epsilon is expressed to a similar extent in most rat tissues examined with the exception of testis, where its expression is approx, three-fold greater. We have also isolated and sequenced the coding and 5'-flanking region of the rat eIF-2B epsilon gene. The 16 exons encoding rat eIF-2B epsilon are contained within 9.5 kb of genomic DNA. Examination of the promoter region of the gene reveals a consensus binding site for the alpha-Pal transcription factor as well as possible cytokine-response elements and binding sites for testis-specific transcription factors.

Amino Acid Sequence↗