Browsing by Author "Johnson, Derrick E."

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    Calcium/Calmodulin-Dependent Protein Kinase II Regulation of IKs during Sustained Beta-Adrenergic Receptor Stimulation
    (Elsevier, 2018) Shugg, Tyler; Johnson, Derrick E.; Shao, Minghai; Lai, Xianyin; Witzmann, Frank; Cummins, Theodore R.; Rubart-Von der Lohe, Michael; Hudmon, Andy; Overholser, Brian R.; Biochemistry and Molecular Biology, School of Medicine
    Background Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. Objective The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. Methods KCNQ1 phosphorylation was assessed using LCMS/MS after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Results Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P = .04) but not at T482 (P = .17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P < .01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P < .01) but not in the dephosphorylated S484 mimic (P = .99). Conclusion CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.
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    Constitutive regulation of the glutamate/aspartate transporter EAAT1 by Calcium-Calmodulin-Dependent Protein Kinase II
    (Wiley, 2017-02) Chawla, Aarti R.; Johnson, Derrick E.; Zybura, Agnes S.; Leeds, Benjamin P.; Nelson, Ross M.; Hudmon, Andy; Biochemistry and Molecular Biology, School of Medicine
    Glutamate clearance by astrocytes is an essential part of normal excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1, respectively, in rodents). While the inhibition of calcium/calmodulin-dependent kinase (CaMKII), a ubiquitously expressed serine/threonine protein kinase, results in diminished glutamate uptake in cultured primary rodent astrocytes (Ashpole et al. 2013), the molecular mechanism underlying this regulation is unknown. Here, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII (using KN-93 or tat-CN21) reduces [3 H]-glutamate uptake in EAAT1 without altering EAAT2-mediated glutamate uptake. While over-expressing the Thr287Asp mutant to enhance autonomous CaMKII activity had no effect on either EAAT1 or EAAT2-mediated glutamate uptake, over-expressing a dominant-negative version of CaMKII (Asp136Asn) diminished EAAT1 glutamate uptake. SPOTS peptide arrays and recombinant glutathione S-transferase-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. Our study is the first to identify a glutamate transporter as a direct CaMKII substrate and suggests that CaMKII signaling is a critical driver of constitutive glutamate uptake by EAAT1.
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    Mitochondrial Ca2+ Uniporter and CaMKII in heart
    (Nature Publishing Group, 2014-09-25) Fieni, Francesca; Johnson, Derrick E.; Hudmon, Andy; Kirichok, Yuriy; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The influx of cytosolic Ca2+ into mitochondria is mediated primarily by the mitochondrial calcium uniporter (MCU), a small-conductance, Ca2+-selective channel-. MCU modulates intracellular Ca2+ transients and regulates ATP production and cell death. Recently, Joiner et al. reported that MCU is regulated by mitochondrial CaMKII, and this regulation determines stress response in heart. They reported a very large current putatively mediated by MCU that was about two orders of magnitude greater than the MCU current (IMCU) that we previously measured in heart mitochondria. Also, the current traces presented by Joiner et al. showed unusually high fluctuations incompatible with the low single-channel conductance of MCU. Here we performed patch-clamp recordings from mouse heart mitochondria under the exact conditions used by Joiner et al. We confirmed that IMCU in cardiomyocytes is very small and showed that it is not directly regulated by CaMKII. Thus the currents presented by Joiner et al. do not correspond to MCU, and there is no direct electrophysiological evidence that CaMKII regulates MCU.
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    Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling
    (Nature Publishing Group, 2016) Zhou, Chaoming; Ramaswamy, Swarna S.; Johnson, Derrick E.; Vitturi, Dario A.; Schopfer, Franciso J.; Freeman, Bruce A.; Hudmon, Andy; Levitan, Edwin S.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) oxidation controls excitability and viability. While hydrogen peroxide (H2O2) affects Ca(2+)-activated CaMKII in vitro, Angiotensin II (Ang II)-induced CaMKIIδ signaling in cardiomyocytes is Ca(2+) independent and requires NADPH oxidase-derived superoxide, but not its dismutation product H2O2. To better define the biological regulation of CaMKII activation and signaling by Ang II, we evaluated the potential for peroxynitrite (ONOO(-)) to mediate CaMKII activation and downstream Kv4.3 channel mRNA destabilization by Ang II. In vitro experiments show that ONOO(-) oxidizes and modestly activates pure CaMKII in the absence of Ca(2+)/CaM. Remarkably, this apokinase stimulation persists after mutating known oxidation targets (M281, M282, C290), suggesting a novel mechanism for increasing baseline Ca(2+)-independent CaMKII activity. The role of ONOO(-) in cardiac and neuronal responses to Ang II was then tested by scavenging ONOO(-) and preventing its formation by inhibiting nitric oxide synthase. Both treatments blocked Ang II effects on Kv4.3, tyrosine nitration and CaMKIIδ oxidation and activation. Together, these data show that ONOO(-) participates in Ang II-CaMKII signaling. The requirement for ONOO(-) in transducing Ang II signaling identifies ONOO(-), which has been viewed as a reactive damaging byproduct of superoxide and nitric oxide, as a mediator of GPCR-CaMKII signaling.
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    Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress
    (Public Library of Science (PLoS), 2015) Gaji, Rajshekhar Y.; Johnson, Derrick E.; Treeck, Moritz; Wang, Mu; Hudmon, Andy; Arrizabalaga, Gustavo; Department of Pharmacology and Toxicology, IU School of Medicine
    Members of the family of calcium dependent protein kinases (CDPK's) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii.