Biomedical subjects

Justin A MacDonald

Publications and source records attributed to Justin A MacDonald.

17 recordsLinked to original sources

PubMed2007

Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide.

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca(2+)-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent K(i) value of 3.4 microM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues.

Amino Acid Sequence↗

PubMed2006

Identification and characterization of D-AKAP1 as a major adipocyte PKA and PP1 binding protein.

Protein kinase A (PKA) plays an important role in the regulation of lipid metabolism in adipocytes. The activity of PKA is known to be modulated by its specific location in the cell, a process mediated by A-kinase anchoring proteins (AKAPs). In order to examine the subcellular localization of PKA in this tissue we performed a search for AKAP proteins in adipocytes. We purified a 120 kDa protein which can bind both the regulatory subunit of PKA as well as the catalytic subunit of protein phosphatase 1 (PP1). This protein was found to be enriched in the lipid droplet fraction of primary adipocytes and was identified as D-AKAP1. This protein may play an important role in the regulation of PKA in adipocytes.

A Kinase Anchor Proteins↗

PubMed2006

Identification of a 115kDa MAP-kinase activated by freezing and anoxic stresses in the marine periwinkle, Littorina littorea.

The mitogen-activated protein kinase (MAPK) cascade regulates changes in gene transcription by transmitting extracellular stimuli from the plasma membrane to the cell nucleus and has an important role to play in organismal responses to environmental stresses. The activities of MAPKs were investigated in the marine gastropod mollusk, Littorina littorea, a species that tolerates both extracellular freezing and long term oxygen deprivation. In-gel kinase assays revealed the presence of two MAPKs in foot muscle and hepatopancreas, a 42 and a 115kDa protein. Immunoblot analysis showed that both were MAPK proteins and that one was the periwinkle homologue of p42(ERK2). Size exclusion chromatography confirmed the 115kDa size of the novel snail MAPK and its role as the dominant MAPK activity in foot muscle. In-gel kinase assays, immunoblotting with phospho-specific ERK antibody, as well as kinase activity profiles from hydroxyapatite chromatography demonstrated that p115 MAPK kinase activity was increased in foot muscle in response to in vivo freezing or anoxia exposures. The results suggest a role for this novel kinase in environmental stress response.

Animals↗

PubMed2006

Spatial audio through a bone conduction interface.

Headphones are the standard presentation device for radio communication in the military. Although bone conduction devices possess several advantages over headphones for some military applications, they are generally considered inappropriate for inclusion in a multi-channel system. The current study tested the feasibility of a multi-channel bone conduction system by measuring the localizability of spatialized auditory stimuli presented through a pair of bone conduction vibrators. Listeners localized a Gaussian noise stimulus spatialized with individualized head-related transfer functions (HRTFs). The sounds were presented from eight virtual locations on the horizontal plane (0, +/-45, +/-90, +/-135, and 180 degrees ) through either stereo headphones or a stereo bone conduction system. Localization performance was found to be nearly identical for both audio systems, indicating that bone conduction systems can be effectively used for displaying spatial information.

Acoustic Stimulation↗

PubMed2005

Mitogen-activated protein kinases and selected downstream targets display organ-specific responses in the hibernating ground squirrel.

The responses of mitogen-activated protein kinase (MAPK) family members, including the extracellular signal-regulated kinases (ERKs), the c-jun NH2-terminal kinases (JNKs) and p38MAPK, during mammalian hibernation were analyzed in five organs of Richardson's ground squirrels, Spermophilus richardsonii. Each kinase subfamily responded differently in torpor and each showed organ-specific patterns of response. ERK1/2 activities increased significantly in muscle and brain during hibernation but decreased in kidney and liver. JNK activity rose in four organs (except brain) during hibernation whereas active, phosphorylated p38MAPK increased only in muscle and heart. Activities of ERK-activated kinases also responded to hibernation: MAPKAPK-1 rose in muscle and brain, MAPKAPK-2 decreased in liver and kidney but rose in the other three organs, and p70S6K kinase activity decreased kidney and heart. Transcription factors, c-Jun and CREB, also showed organ-specific responses during torpor. The data suggest key roles for MAPKs in the regulation of the known organ-specific changes in gene expression and protein phosphorylation that define the hibernation phenotype.

Animals↗

PubMed2004

Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins.

Cyclic nucleotides acting through their associated protein kinases, the cGMP- and cAMP-dependent protein kinases, can relax smooth muscles without a change in free intracellular calcium concentration ([Ca2+]i), a phenomenon referred to as Ca2+ desensitization. The molecular mechanisms by which these kinases bring about Ca2+ desensitization are unknown and an understanding of this phenomenon may lead to better therapies for treating diseases involving defects in the contractile response of smooth muscles such as hypertension, bronchospasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Utilizing a combination of real-time proteomics and smooth muscle physiology, we characterized a distinct subset of protein targets for cGMP-dependent protein kinase in smooth muscle. Among those phosphoproteins identified was calponin homology-associated smooth muscle (CHASM), a novel protein that contains a calponin homology domain and shares sequence similarity with the smoothelin family of smooth muscle specific proteins. Recombinant CHASM was found to evoke relaxation in a concentration dependent manner when added to permeabilized smooth muscle. A co-sedimentation assay with actin demonstrated that CHASM does not possess actin binding activity. Our findings indicate that CHASM is a novel member of the smoothelin protein family that elicits Ca2+ desensitization in smooth muscle.

Actins↗

PubMed2004

Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides.

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.

Animals↗

PubMed2004

C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats.

The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5. A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern. We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3'-end formation. Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5. We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2. When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD. We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates.

Amino Acid Sequence↗

PubMed2004

Improving upon the in vitro biological activity of antithrombotic disulfides.

Several sulfur-containing compounds, isolated from garlic, have been implicated as highly active antithrombotic agents. We have prepared 10 new aromatic disulfides and an aromatic thiosulfonate in order to determine the in vitro response of human platelets to dosages of these compounds. The poor biological activity of PhSSCH3 was enhanced by the introduction of, inter alia, a nitro group onto the aromatic ring. The nitro group increased potency by activating the disulfide linkage. Anti-platelet aggregation activity was also enhanced by increasing the lipophilicity of one test compound. The ability of an aromatic disulfide to inhibit platelet aggregation can be enhanced by appending an electron-withdrawing group to the aromatic ring. The results presented establish that the aromatic thiosulfonate is a very effective inhibitor of platelet aggregation.

Disulfides↗

PubMed2003

Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences.

The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.

Amino Acid Sequence↗

PubMed2003

Is the area measure a historical anomaly?

Green's well-known area theorem establishes an equivalence between the area under the yes-no ROC curve and the percent correct of an unbiased observer in a two-alternative forced-choice (2AFC) task with equivalent stimuli. In this article, we show that this conversion from yes-no detection data to hypothetical performance in a 2AFC task is unnecessary: The same yes-no detection data that are used to compute the area statistic can always be used to compute the percent correct of an unbiased observer in the yes-no detection task itself. We also show that the ROC curve may not be the ideal graphical device for many investigators. A more natural representation of the difficulty of a discrimination task is obtained by plotting the distribution of the posterior betting odds under equal base rates, which can be estimated from their distributions under unequal base rates. Finally, unlike the area measure and other traditional detection theory statistics, both the yes-no percent correct measure and the odds distributions generalize in an obvious and direct way to classification paradigms with more than two responses (e.g., identification).

History↗

PubMed2002

Purification and characterization of fructose bisphosphate aldolase from the ground squirrel, Spermophilus lateralis: enzyme role in mammalian hibernation.

Fructose-1,6-bisphosphate (F1,6P(2)) aldolase was purified to homogeneity from skeletal muscle of the golden-mantled ground squirrel, Spermophilus lateralis. Enzyme properties were examined at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C); parallel studies assessed rabbit muscle aldolase for comparison. Kinetic properties of each enzyme were differentially affected by assay temperature. For example, the K(m) for F1,6P(2) of ground squirrel aldolase was 0.9+/-0.05 microM at 37 degrees C and 50% higher (1.45+/-0.04 microM) at 5 degrees C, whereas the K(m) of rabbit aldolase increased threefold over the same temperature range. The inhibitory effects of adenylates were similar at both temperatures for the ground squirrel enzyme, but inhibition by adenosine 5(')-diphosphate, adenosine 5(')-monophosphate, and inosine 5(')-monophosphate was substantially reduced at 5 degrees C for rabbit aldolase. Inhibition by inorganic phosphate increased at lower temperatures for both enzymes; for ground squirrel aldolase, the K(i) was 1.18+/-0.1mM at 37 degrees C and 0.23+/-0.05 mM at 5 degrees C. Inhibition of aldolase by inorganic phosphate could be one factor that helps to shut down glycolysis during hibernation. Thus, mammalian hibernators may exploit low-temperature characteristics of aldolase to benefit the metabolic needs of the hibernating state.

Adenosine Diphosphate↗

PubMed2002

Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase.

A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr(709), and two other sites at Thr(695) and Thr(495). One of the sites for cAMP-dependent protein kinase (PKA) was Ser(694). Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr(695)-->Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr(695) was the major inhibitory site, Thr(709) had only a slight inhibitory effect and Ser(694) had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.

Amino Acid Sequence↗

PubMed2002

Smooth muscle myosin phosphatase-associated kinase induces Ca2+ sensitization via myosin phosphatase inhibition.

Smooth muscle calcium sensitization reflects an inhibition of myosin light chain phosphatase (SMPP-1m) activity; however, the underlying mechanisms are not well understood. SMPP-1m activity can be modulated through phosphorylation of the myosin targeting subunit (MYPT1) by the endogenous myosin phosphatase-associated kinase, MYPT1 kinase (MacDonald, J. A., Borman, M. A., Muranyi, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419-2424). Recombinant chicken gizzard MYPT1 (M130) was phosphorylated in vitro by a recombinant MYPT1 kinase, and the sites of phosphorylation were identified as Thr(654), Ser(808), and Thr(675). Introduction of recombinant MYPT1 kinase elicited a calcium-independent contraction in beta-escin-permeabilized rabbit ileal smooth muscle. Using an antibody that specifically recognizes MYPT1 phosphorylated at Thr(654) (M130 numbering), we determined that this calcium-independent contraction was correlated with an increase in MYPT1 phosphorylation. These results indicate that SMPP-1m phosphorylation by MYPT1 kinase is a mechanism of smooth muscle calcium sensitization.

Amino Acid Sequence↗

PubMed2002

Protein phosphatase type-1 from skeletal muscle of the freeze-tolerant wood frog.

We evaluated the effects of freezing, dehydration and anoxia stresses on muscle PP-1 activity in the freeze-tolerant amphibian, Rana sylvatica. In addition, PP-1 catalytic subunit (PP-1c) was purified to homogeneity to assess the biochemical properties of the enzyme from a freeze-tolerant vertebrate. Freezing stimulated a rise in the amount of active PP-1 (70% above the control) at 20 min post-nucleation. With longer freezing (1-12 h), the amount of active enzyme returned to control levels, and the amount of total PP-1 fell, decreasing by up to 43%. This decline in total PP-1 kept the % active at a high value throughout the freeze. Anoxia exposure (12 h) reduced the active PP-1 by 60%, but had no effect on total PP-1 activity. Neither dehydration nor rehydration had any significant effect on the amounts of either total or active PP-1. PP-1 activity associated with the myofibril fraction increased, while activity associated with the glycogen pellet decreased in response to freezing and dehydration, but not anoxia. Purified frog PP-1c showed a variety of properties that are typical of the enzyme from other sources. In addition, the enzyme was strongly inhibited by AMP and weakly by ADP and ATP; the physiological relevance of inhibition by nucleotides remains to be determined. Overall, the results suggest an important role for PP-1 in signal transduction in the skeletal muscle of freeze-tolerant amphibians.

Adaptation, Physiological↗

PubMed2002

A strategy for the rapid identification of phosphorylation sites in the phosphoproteome.

Edman phosphate ((32)P) release sequencing provides a high sensitivity means of identifying phosphorylation sites in proteins that complements mass spectrometry techniques. We have developed a bioinformatic assessment tool, the cleavage of radiolabeled protein (CRP) program, which enables experimental identification of phosphorylation sites via (32)P labeling and Edman degradation of cleaved proteins obtained at femtomole levels. By observing the Edman cycle(s) in which radioactivity is found, candidate phosphorylation sites are identified by determining which residues occur at the observed number of cycles downstream from a peptide cleavage site. In cases where more than one residue could be responsible for the observed radioactivity, additional experiments with cleavage reagents having alternative specificities may resolve the ambiguity. Given a protein sequence and a cleavage site, CRP performs these experiments in silico, identifying resolved sites based on user-supplied experimental data, as well as suggesting combinations of reagents for additional analyses. Analysis of the PhosphoBase protein sequence database suggests that CRP data from two cleavage experiments can be used to identify unambiguously 60% of known phosphorylation sites. Data from additional cleavage experiments may increase the overall coverage to 70% of known sites. By comparing theoretical data obtained from the CRP program with (32)P release data obtained from an Edman sequencer, a known phosphorylation site was identified unambiguously and correctly. In addition, our results show that in vivo phosphorylation sites can be determined routinely by differential proteolysis analysis and Edman cycling with less than 1 fmol of protein and 1000 cpm.

Amino Acid Sequence↗

PubMed2002

Intelligibility of speech in a virtual 3-D environment.

In a simulated air traffic control task, improvement in the detection of auditory warnings when using virtual 3-D audio depended on the spatial configuration of the sounds. Performance improved substantially when two of four sources were placed to the left and the remaining two were placed to the right of the participant. Surprisingly, little or no benefits were observed for configurations involving the elevation or transverse (front/back) dimensions of virtual space, suggesting that position on the interaural (left/right) axis is the crucial factor to consider in auditory display design. The relative importance of interaural spacing effects was corroborated in a second, free-field (real space) experiment. Two additional experiments showed that (a) positioning signals to the side of the listener is superior to placing them in front even when two sounds are presented in the same location, and (b) the optimal distance on the interaural axis varies with the amplitude of the sounds. These results are well predicted by the behavior of an ideal observer under the different display conditions. This suggests that guidelines for auditory display design that allow for effective perception of speech information can be developed from an analysis of the physical sound patterns.

Accidents, Aviation↗