Charles FB Holmes | University of Alberta (original) (raw)

Papers by Charles FB Holmes

Journal of Biological Chemistry, 1993

In this paper, we report that ~3 4 '~'~ protein kinase phosphorylates recombinant fragments of sk... more In this paper, we report that ~3 4 '~'~ protein kinase phosphorylates recombinant fragments of skeletal muscle dystrophin with a maximal incorporation of 1.8 mol of PJmol of protein. Phosphorylation of both serine and threonine residues occurs within the carboxylterminal 201 amino acids of dystrophin, with phosphothreonine localized to within 25 residues of the carboxyl terminus. Supporting these in vitro studies, we also show that native dystrophin is phosphorylated by ~3 4~~'~ kinase in isolated sarcolemmal vesicles. Sequence analysis indicates two consensus sites for p34*" protein kinase within the carboxyl-terminal 201 amino acids of dystrophin. Importantly, neither of these sites is conserved in dystrophin-related protein, and only one site is conserved in the 71-kDa alternative product of the Duchenne muscular dystrophy gene, despite an otherwise extremely high degree of sequence conservation between these proteins. Importantly, in this study we also show that dystrophin is phosphorylated in vivo in rat skeletal muscle primary cultures, and we suggest that further investigation of both in vivo and in vitro phosphorylation of this protein will comprise an important part in determination of its function(s). Dystrophin is the protein product of the Duchenne muscular dystrophy (DMD)' gene, which is absent in muscle from DMD patients (Ahn and Kunkel, 1993). The function of this protein is not known. In skeletal muscle, dystrophin is a major component of the subsarcolemmal cytoskeleton (Ohlendieck and Campbell, 1991) where it is associated with a complex of * This work was supported by the Muscular Dystrophy Association of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Journal of Biological Chemistry, 2019

Journal of Molecular Biology, 2006

International Journal of Cancer, 1999

The regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundame... more The regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundamental roles these zinc-endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP-2 contains 29 potential phosphorylation sites. Mass spectrometry reveals that at least five of these sites are phosphorylated in hrMMP-2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP-2 immunoreactivity on 2D immunoblots consistent with phosphorylation, and purified MMP-2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP-2 from HT1080 cell-conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP-2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP-2 significantly affects its enzymatic properties. Consistent with this, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase significantly increases its activity. We conclude that MMP-2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.

Chemical Research in Toxicology, 1997

Nucleic Acids Research, 1990

Journal of Virology, 2003

Rubella virus is an enveloped positive-strand RNA virus of the family Togaviridae . Virions are c... more Rubella virus is an enveloped positive-strand RNA virus of the family Togaviridae . Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the R...

Journal of Biological Chemistry, 2001

Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processe... more Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 Å. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive ␤12-␤13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and-2A but not PP-2B (calcineurin) may be reassessed in light of this study.

Journal of Biological Chemistry, 2007

TIMAP (TGF-␤1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predomina... more TIMAP (TGF-␤1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [ 32 P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3␤. Site-specific Ser to Ala substitution identified amino acid residues Ser 333 /Ser 337 as the likely PKA/GSK-3␤ phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAP V64A/F66A) abolished PP1c binding and TIMAPassociated PP1c activity. TIMAP V64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3␤ stimulated phosphorylation of TIMAP V64A/F66A , but not wild-type TIMAP, suggesting that the PKA/GSK-3␤ site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3␤-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.

Journal of Biological Chemistry, 2014

Background: Lipin-1 functions as a phosphatidate phosphatase in glycerolipid synthesis and as a c... more Background: Lipin-1 functions as a phosphatidate phosphatase in glycerolipid synthesis and as a co-transcriptional regulator. Results: Lipin-1 contains conserved N-terminal motifs, which when mutated decrease phosphatase activity, nuclear localization, and binding to protein phosphatase-1c␥. Conclusion: The lipin-1 N-terminal domain is important in regulating its activities. Significance: Lipin-1 binds to protein phosphatase-1c␥ through its N-terminal domain, and this potentially regulates lipin-1 localization and function. Lipin-1 is a phosphatidate phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1c␥ through a similar HVRF binding motif. This interaction depends on Mg 2؉ or Mn 2؉ and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1c␥ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1c␥ binding. PP-1c␥ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1c␥ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and phosphatidate phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1c␥.

Journal of Biological Chemistry, 1997

The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful ... more The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful liver tumor promoters and potent inhibitors of the catalytic subunits of protein phosphatase-1 and-2A (PP-1c and PP-2Ac). In marked contrast to microcystins, which interact covalently with PP-1 and PP-2A, the nodularins do not bind covalently to PP-1 and PP-2A and may additionally possess unique carcinogenic properties. The conformation of microcystin-LR has been determined in solution and bound to PP-1c. We show here that the free NMR solution structures of two distinct microcystin structural congeners (microcystin-LR and-LL) are remarkably similar to the bound crystal structure of microcystin-LR. We have exploited this finding by using Metropolis Monte Carlo modeling to dock the solution structures of microcystin-LL and the marine toxin motuporin (nodularin-V) onto the crystal structure of PP-1c. Both of these toxins occupy a position similar to that of microcystin-LR when bound to PP-1c. However, although there are relatively minor differences in the structural orientation of microcystin-LL compared with microcystin-LR, there is a striking difference in the position of the N-methyldehydrobutyrine residue in motuporin relative to the comparable N-methyldehydroalanine residue in microcystin-LR. We propose that this difference in orientation provides a molecular explanation for why nodularins are incapable of forming a covalent linkage with PP-1c. Furthermore, the predicted position of N-methyldehydrobutyrine in motuporin is at the surface of the PP-1c-toxin complex, which may thus facilitate chemical interaction with a further macromolecule(s) possibly relating to its carcinogenic properties. PP-1c and PP-2Ac are also targets for other marine toxins such as okadaic acid and calyculin A. It was therefore of interest to use Metropolis Monte Carlo modeling to dock the known free crystal structures of okadaic acid and calyculin A to the crystal structure of PP-1c. These experiments predict that both okadaic acid and calyculin A are strikingly similar to microcystins and motuporin in their tertiary structure and relative PP-1c binding position.

Journal of Biological Chemistry, 2000

Site-directed mutagenesis was used to investigate the mechanism of interaction between the cataly... more Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1c␥) and members of the calyculin family of toxins. Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group. We provide experimental evidence implicating Tyr-134 as an important residue in PP-1c␥ that mediates interactions with the calyculins. Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1c␥ to clavosines A and B and calyculin A. In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors. The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase. Clavosine B inhibited PP-1c␥ Y134D with a 310-fold decrease in potency. Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant. These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase. The clavosines and calyculin A were tested for their ability to inhibit other mutants of PP-1c␥ (including Ile-133, Val-223, and Cys-291). Our mutagenesis studies provide an experimental basis for assessing models of calyculin binding found in the literature (Lindvall, M. K., Pihko,

Journal of Biological Chemistry, 2012

Background: Heterozygous mutations in the cytoplasmic domain of phospholamban cause lethal dilate... more Background: Heterozygous mutations in the cytoplasmic domain of phospholamban cause lethal dilated cardiomyopathy. Results: The mutations alter phospholamban-protein kinase A interactions that are essential for substrate recognition and phosphorylation. Conclusion: Hereditary mutations in phospholamban that prevent phosphorylation by protein kinase A will lead to chronic inhibition of SERCA. Significance: Arginines in the cytoplasmic domain of phospholamban should be considered hot spots for hereditary mutations leading to dilated cardiomyopathy.

Frontiers in Bioscience, 1999

Introduction 3. Okadaic acid 3.1. Diarrhetic shellfish poisoning 3.2. Dinoflagellates 3.3. Okadai... more Introduction 3. Okadaic acid 3.1. Diarrhetic shellfish poisoning 3.2. Dinoflagellates 3.3. Okadaic acid as a potent inhibitor of protein phosphatases 4. Microcystins 4.1. Barcoo fever and cyanobacteria 4.2. Microcystin structure and phosphatase inhibition 5. Molecular interactions 5.1. Insights from structural analysis of the marine toxins 5.1.1. Okadaic acid 5.1.2. Microcystins 5.2. Insights from sequence comparisons of PP-1c and PP-2Ac 5.3. Insights from the crystal structure of microcystin-LR bound to PP-1c. 5.3.1. Interactions with catalytic metals 5.3.2. Interactions with the L7 loop 5.3.3. Hydrophobic interactions 5.4. Insights from mutagenesis studies 6. Molecular modeling of okadaic acid bound to PP-1c: a common marine toxin binding site 7. Comparison with PP-2B (calcineurin) and novel phosphatases 8. Perspective 9. Acknowledgments 10. References

FEBS Letters, 1987

FEBS Lett 204,61-66] have shown that phosphoserine can be converted to S-ethylcysteine by p-elimi... more FEBS Lett 204,61-66] have shown that phosphoserine can be converted to S-ethylcysteine by p-elimination and addition of ethanethiol. I have utilised this modification to develop a rapid method for the selective purification of phosphoserine-containing peptides from complex mixtures. Changing phosphoserine to Sethylcysteine increases the hydrophobicity of a peptide, altering its mobility during reversephase chromatography. The number of S-ethylcysteine residues in a peptide can be quantified at the picomolar level, following acid hydrolysis and conversion to the phenylthiocarbamyl derivative. The procedure may be particularly powerful for the analysis of peptides that are phosphorylated at multiple sites in vivo.

FEBS Letters, 1990

Acanthifolicin (9,!0-epithio-okadaic acid from Pundoras acunthifolium) inhibited protein phosphat... more Acanthifolicin (9,!0-epithio-okadaic acid from Pundoras acunthifolium) inhibited protein phosphatase-1 (PP!) similarly to okadaic acid (IC,, =20 nM and 19 nM, respectively) but was slightly less active against protein phosphatase-2A (PPZA) (IC,, = 1 nM and 0.2 nM, respectively). Methyl esterification of acanthifolicin sharply reduced its activity. PPZA was inhibited with an IC,, = 5.0 FM, whilst PP! was inhibited < 10% at 250 FM toxin. Okadaic acid methyl ester was similarly inactive whereas dinophysistoxin-1 (35-methyl okadaic acid) inhibited PPl/2A almost as potently as okadaic acid. Pure acanthifolicin/okadaic acid methyl ester may be useful as specific inhibitors of PP2A at l-10 PM concentrations in vitro and perhaps in vivo. The data also indicate that a region on these toxins important for PPl/ZA inhibition comprises the single carboxyl group.

FEBS Letters, 1993

The unicellular marine dinoflagellate, Prorocentrum lima, an established producer of okadaic acid... more The unicellular marine dinoflagellate, Prorocentrum lima, an established producer of okadaic acid (OA), was shown to contain a type-1 protein phosphatase (PP-1) the biochemical profile of which on Mono-Q and Superdex-75 fast protein liquid chromatography was identical to the catalytic subunit of PP-1 from rabbit skeletal muscle. Purified P lima PP-1 (apparent molecular mass 37.5 kDa) was highly sensitive to inhibition by mammalian protein phosphatase inhibitor-1 and inhibitor-2, and to OA itself. A 6-7-fold increase in OA production by P lima, when grown under controlled conditions, correlated with an up to 300-fold increase in P lima PP-1 activity. Furthermore, P lima did not contain any detectable type-2A protein phosphatase activity. This study represents the first identification of a serine/threonine protein phosphatase in a dinoflagellate.

Environmental Science & Technology, 1994

Canadian Journal of Fisheries and Aquatic Sciences, 1996

Micmcystin-LR (MC-LR) wncen&&ns were examined in water, phytoplankton, invertebrates, and two fis... more Micmcystin-LR (MC-LR) wncen&&ns were examined in water, phytoplankton, invertebrates, and two fishes for up to 3 years in four cmtral Alberta lakes spanning a tmphic gradient in total phosphorus from I5 to over 500 pgL-' in epilinmetic waters MC-LR was not detected by HPLC in phytoplankton tinm an oligo-mesotmphic lake, was less than 150 ng cellular totiml-' of lake water in a eutmphic-bypereatmphic lake, and was up to M)(H) and I I 000 ngL-' in two hypenubophic lakes. MC-LR in phytoplankton was strongly correlated with the abundance of the cyanobacterium Micm~stir oeruginosa, and with aqueous micmcystin concsntration. determined by protein phosphatase bioassay (r = 0.83). MC-LR was also detected in moplankton (up to 67 pgg-' ofbiomass) and MC-LR concenti~ in zooplankton and~phytoptankton were correlated (r = 0.69). Although nine groups of macroinvertebrates were analyzed, MC-LR was only detected in gastmpods (up to 120 pgg-'1. MC-LR appears te be bansferred to invertebrates through grazing activity. MC-LR was not detected in the liven ofnmthem pike (Esox luciw) and white sucker (CawfomuE mmmermni) ~llectcd from one lake containing toxinproducing phymplankton. Accumulation of MC-LR in aquatic fwd webs appears to-in the primary consumer with probable tmnsfer of the toxin to higher trophic levels. Rhm6 : Pendant UM p&ode atteigmnt une d& de 3 ans, on a mesurl les concentrations de micrwystine-LX (MC-LR) dam l'eau et le phytoptancton ainsi que chez tes inv&btis et chek dew esp&s de poissons de 4 lacs du centre de I'Alber+a 06 le gradient tmphique de phosphors total Cpilim&tique allait de 15 B phrs de 500 pgL-'. La HPLC du phytoplancton du tat oIigom6sotmphe n'a pas mis en tvidence de MC-LR; la concentration de la toxine allulaire etait inf&ieae g 150 ng.L-' d'eau dam le lac eutrophe-hypereutmphe et elk atteignait 6000 et I1 000 ngL-' dam Ies deux hypereutmphes. Par aillew, on a not6 une forte cotitation entre la concentration de MC-LR phytoplmctcmique et t'abor&xe de la cyan&&de Micmcystir ~eruginma et la concenbaticnrde micnxystine aquewse, do&e avec une pmt6ine-phospbatas.c (r= 0,83). Dn a aussi d6tectC de la MC-LR chez le moplancton (jusqu? 67 pgg-' de biomassc) et observt une con&tion entre Ia concentrations de MC-LR zwptanctonique et phytoplmctonique (r = 0.69). On a anaIys& neufgmupes de macroiwe&&s. mais la MC-LR n'a 6tC detect6 que chez les gast6mpodes (jusqu'& I20 vgg-'). La MC-LR semble passeraux invert&b& par le tmutage. On "'en a pas d6tectt dam le foie du bmche @ox /ucim) et du meunier noir (Caxmmw commermnz]~r&v6s dam l'un des lacs oti le phytoplaneton est pmdwteti de toxine. La bioconcentation de MC-LR dans lea r6seaux tmphiques aquatiques sable survenir au niveau des consommatews primaires et il y a probablement tmnsfen de la toxine auxnivcaux tmphiques plus tleds. frmduit par la R&ction]

Canadian Journal of Fisheries and Aquatic Sciences, 1997

Freshwater clams (Anodonta grandis simpsoniana) exposed to 51-55 µg · L-1 of dissolved microcysti... more Freshwater clams (Anodonta grandis simpsoniana) exposed to 51-55 µg · L-1 of dissolved microcystin-LR (MC-LR) in the laboratory for 3 days did not accumulate MC-LR equivalents (MC-LReq). However, clams placed in three eutrophic lakes with phytoplankton containing MC-LR (concentrations from below detection to 8.3 µg · L-1 cellular toxin) for 12-28 days accumulated the toxin (24 ± 7 to 527 ± 330 ng · g-1 MC-LReq; mean ± SE). The relative MC-LReq concentrations in clams reflected MC-LR concentrations in lake phytoplankton, but individual variation was high. In individual clams exposed for 24 days, the average MC-LReq concentration was usually greater in the visceral mass than in gills and muscle, but average toxin concentrations in the three tissues were similar (587, 310, and 364 ng · g dry weight-1). In clams removed from the lake and placed in toxin-free water, MC-LReq concentrations in tissues declined rapidly for 6 days (by 69-88%) but remained relatively stable for the remaining ...