Derrick E. Johnson

A soluble α-synuclein construct forms a dynamic tetramer

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated “hairpin” structure of α-synuclein.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites.

Background: CaMKII is up-regulated in heart failure and modulates Na+ current (INa), yet the mechanism is unclear. Result: CaMKII phosphorylates several sites in the first intracellular loop of NaV1.5, thereby altering INa gating properties. Conclusion: This multisite phosphorylation may contribute to acquired arrhythmogenesis. Significance: Identification of these regulatory sites is critical for potential therapeutic targeting of CaMKII and NaV1.5 in failing hearts. The cardiac Na+ channel NaV1.5 current (INa) is critical to cardiac excitability, and altered INa gating has been implicated in genetic and acquired arrhythmias. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause INa gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on NaV1.5 that mediate(s) the CaMKII-induced alterations in INa gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of NaV1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δC-CaMKII and the intracellular loop between domains 1 and 2 of NaV1.5 was observed. This region was also phosphorylated by δC-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNaV1.5 were co-expressed with GFP-δC-CaMKII in HEK293 cells, and INa was recorded. As observed in myocytes, CaMKII shifted WT INa availability to a more negative membrane potential and enhanced accumulation of INa into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted INa availability without the need for CaMKII. CaMKII-dependent phosphorylation of NaV1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.

Serum deprivation inhibits the transcriptional co-activator YAP and cell growth via phosphorylation of the 130-kDa isoform of Angiomotin by the LATS1/2 protein kinases

Significance This study defines a unique mechanism controlling the activation of Hippo signaling and consequent inhibition of cell growth. Specifically, serum starvation is found to induce the large tumor suppressor (LATS)1/2 kinases to phosphorylate and thus stabilize the 130 kDa isoform of the membrane-associated polarity protein angiomotin (Amot130). As a consequence, Amot130 recruits the E3 protein-ubiquitin ligase atrophin-1 interacting protein 4. This multiprotein complex then signals the degradation of Yes-associated protein (YAP) and the inhibition of cell growth. These findings significantly modify our current view that YAP phosphorylation by LATS1/2 is sufficient for its inhibition in mammals and thus for growth arrest. Large tumor suppressor (LATS)1/2 protein kinases transmit Hippo signaling in response to intercellular contacts and serum levels to limit cell growth via the inhibition of Yes-associated protein (YAP). Here low serum and high LATS1 activity are found to enhance the levels of the 130-kDa isoform of angiomotin (Amot130) through phosphorylation by LATS1/2 at serine 175, which then forms a binding site for 14-3-3. Such phosphorylation, in turn, enables the ubiquitin ligase atrophin-1 interacting protein (AIP)4 to bind, ubiquitinate, and stabilize Amot130. Consistently, the Amot130 (S175A) mutant, which lacks LATS phosphorylation, bound AIP4 poorly under all conditions and showed reduced stability. Amot130 and AIP4 also promoted the ubiquitination and degradation of YAP in response to serum starvation, unlike Amot130 (S175A). Moreover, silencing Amot130 expression blocked LATS1 from inhibiting the expression of connective tissue growth factor, a YAP-regulated gene. Concordant with phosphorylated Amot130 specifically mediating these effects, wild-type Amot130 selectively induced YAP phosphorylation and reduced transcription of connective tissue growth factor in an AIP4-dependent manner versus Amot130 (S175A). Further, Amot130 but not Amot130 (S175A) strongly inhibited the growth of MDA-MB-468 breast cancer cells. The dominant-negative effects of Amot130 (S175A) on YAP signaling also support that phosphorylated Amot130 transduces Hippo signaling. Likewise, Amot130 expression provoked premature growth arrest during mammary cell acini formation, whereas Amot130 (S175A)-expressing cells formed enlarged and poorly differentiated acini. Taken together, the phosphorylation of Amot130 by LATS is found to be a key feature that enables it to inhibit YAP-dependent signaling and cell growth.

High-throughput characterization of intrinsic disorder in proteins from the Protein Structure Initiative.

The identification of intrinsically disordered proteins (IDPs) among the targets that fail to form satisfactory crystal structures in the Protein Structure Initiative represents a key to reducing the costs and time for determining three-dimensional structures of proteins. To help in this endeavor, several Protein Structure Initiative Centers were asked to send samples of both crystallizable proteins and proteins that failed to crystallize. The abundance of intrinsic disorder in these proteins was evaluated via computational analysis using predictors of natural disordered regions (PONDR®) and the potential cleavage sites and corresponding fragments were determined. Then, the target proteins were analyzed for intrinsic disorder by their resistance to limited proteolysis. The rates of tryptic digestion of sample target proteins were compared to those of lysozyme/myoglobin, apomyoglobin, and α-casein as standards of ordered, partially disordered and completely disordered proteins, respectively. At the next stage, the protein samples were subjected to both far-UV and near-UV circular dichroism (CD) analysis. For most of the samples, a good agreement between CD data, predictions of disorder and the rates of limited tryptic digestion was established. Further experimentation is being performed on a smaller subset of these samples in order to obtain more detailed information on the ordered/disordered nature of the proteins.

Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.

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.

Constitutive Regulation of the Glutamate/Aspartate Transporter EAAT1 by Calcium/Calmodulin‐Dependent Protein Kinase II

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 [3H]‐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.

Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) oxidation controls excitability and viability. While hydrogen peroxide (H2O2) affects Ca2+-activated CaMKII in vitro, Angiotensin II (Ang II)-induced CaMKIIδ signaling in cardiomyocytes is Ca2+ 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 Ca2+/CaM. Remarkably, this apokinase stimulation persists after mutating known oxidation targets (M281, M282, C290), suggesting a novel mechanism for increasing baseline Ca2+-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.

Mechanisms Underlying Cooperativity in CaMKII Autophosphorylation and Substrate Phosphorylation

As a member of the calmodulin-activated kinases, Ca2+/calmodulin dependent protein kinase II (CaMKII) is a serine/threonine kinase coupled to calcium signaling. Unlike other multifunctional CaMK members, CaMKII has a unique dodecameric architecture potentially permitting cooperative forms of autoregulation and substrate phosphorylation. We observed that CaMKII phosphorylation of a peptide derived from the autoregulatory domain (AC-2) displays positive cooperativity (nH=1.8). Another T-site binding peptide substrate derived from S1303 phosphorylation site on NR2B also displayed positive cooperativity (nH=2.1). Surprisingly, a truncated form of CaMKII1-317 monomer also shows this cooperativity towards NR2B and AC-2 peptides (nH=1.6 vs 1.8, respectively). Syntide-2, a traditional substrate peptide lacking T-site interactions, does not exhibit cooperativity in substrate phosphorylation for either monomeric or multimeric CaMKII (nH=1.0; nH=1.1, respectively). These data suggest that the positive cooperativity seen with substrate phosphorylation is unique to T-site site binding substrates and may involve potential allosteric substrate interactions on the catalytic surface. Cooperativity within the holoenzyme occurring between subunits has been predicted based on the fact that Ca2+/CaM binding can induce Thr286 autophosphorylation, an intersubunit intraholenzyme reaction, whereby, neighboring subunits act as both kinase and substrate following coincident Ca2+CaM binding. Using Ca2+/CaM-independent activity (i.e. autonomous) as a measure of Thr286 autophosphorylation, CaM titration experiments revealed that both Ca2+/CaM-dependent and autonomous forms of CaMKII activity were cooperative (nH=2.1 for both) for Ca2+/CaM activation and autophosphorylation. Titrating CaM levels to ratios below 1 per holoenzyme (i.e. 12 CaMKII subunits per holoenzyme) generate submaximal autonomous activity, whereas, CaM levels at a ratio of ∼2 per CaMKII holoenzyme generate maximal autonomous activity. Thus, CaM activation of CaMKII appears to follow a cooperative model whereby neighboring subunits within the holoenzyme preferentially obtain the activator to promote autophosphorylation even in the face of limiting CaM.

Activation State-Dependent Substrate Gating in Ca2+/Calmodulin-Dependent Protein Kinase II

Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+ sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+ signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287 (T286 α isoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287 autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287 autophosphorylation. Our data suggest T287 autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.

The Multimeric Architecture of CaMKII Alters Substrate Regulation of Diffusion-Restricted Environments

Calcium/calmodulin dependent protein kinase II (CaMKII) is a family of multifunctional Ser/Thr kinases that play a key role in calcium signaling in many cell types, including neurons, where it performs both structural and signaling roles in learning and memory. CaMKII exists as a dodecameric holoenzyme. Whether the multimeric nature of the CaMKII holoenzyme also produces unique regulation and substrate interactions is unknown; however, having multiple catalytic subunits in each holoenzyme could afford enhanced substrate and binding partner interactions leading to altered rules for substrate selection and phosphorylation compared to monomeric kinases. Using a peptide-based model of CaMKII substrates, we measured substrate phosphorylation in soluble versus immobilized assays designed to represent the compartmentalized, diffusion-restricted environments in which CaMKII is known to function (such as the post-synaptic density [PSD] in the dendritic boutons of neurons) using both CaMKII and a monomeric form of CaMKII (1-316). Solution kinetics revealed very minimal differences in substrate phosphorylation of various substrates, yet significant changes were observed in phosphorylation profiles of immobilized peptide substrates. Strikingly, whereas monomeric CaMKII phosphorylation correlated proportionally to relative Km values from solution assays, multimeric CaMKII displayed preferential phosphorylation of low-affinity substrates (higher Km) and diminished phosphorylation of high-affinity substrates (lower Km). Subsequent experiments involving the phosphorylation of purified PSDs produced >4-fold global enhancement of phosphorylation by multimeric CaMKII compared to the addition of equal catalytic units of monomer, consistent with findings that many of the CaMKII substrates in the PSD lack a canonical phosphorylation motif and could therefore, as low-affinity substrates, take full advantage of CaMKIIs multimeric structure. Thus, the multimeric architecture of CaMKII may confer a unique and novel mechanism for regulating substrate phosphorylation within diffusion-restricted subcellular compartments, which may underscore CaMKIIs dominant role in synaptic plasticity and learning.