Wen-Hai Chou

A semisynthetic epitope for kinase substrates

The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPγS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester–specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38α MAPK, Erk1, Erk2, Akt1, PKCδ, PKCε, Cdk1/cyclinB, CK1, Cdc5, GSK3β, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.NOTE: In the version of this article initially published online, a sentence was missing a word and did not make sense. The corrected sentence now reads, “Erk2 was immunoprecipitated from each of these cell lines and assayed with A*TPγS analogs; N6-phenethyl ATPγS was a preferred nucleotide substrate for AS Erk2 and was not accepted by wild-type Erk2 (data not shown).” The error has been corrected for all versions of the article.

Identification of a Novel Drosophila Opsin Reveals Specific Patterning of the R7 and R8 Photoreceptor Cells

The function of the compound eye is dependent upon a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant files, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes.

Neutrophil protein kinase Cδ as a mediator of stroke-reperfusion injury

Thrombolysis is widely used to intervene in acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Here we describe studies with mice lacking protein kinase Cδ (PKCδ) showing that absence of this enzyme markedly reduces reperfusion injury following transient ischemia. This was associated with reduced infiltration of peripheral blood neutrophils into infarcted tissue and with impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. Total body irradiation followed by transplantation with bone marrow from PKCδ-null mice donors reduced infarct size and improved neurological outcome in WT mice, whereas marrow transplantation from WT donors increased infarction and worsened neurological scores in PKCδ-null mice. These results indicate an important role for neutrophil PKCδ in reperfusion injury and strongly suggest that PKCδ inhibitors could prove useful in the treatment of stroke.

Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Ethanol enhances γ-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABAA receptors, especially at low concentrations achieved during social drinking. We postulated that the ϵ isoform of protein kinase C (PKCϵ) regulates the ethanol sensitivity of GABAA receptors, as mice lacking PKCϵ show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCϵ mutant to selectively inhibit the catalytic activity of PKCϵ. We used this mutant and PKCϵ-/- mice to determine that PKCϵ phosphorylates γ2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at α1β2γ2 receptors, which is the most abundant GABAA receptor subtype in the brain. Our findings indicate that PKCϵ phosphorylation of γ2 regulates the response of GABAA receptors to specific allosteric modulators, and, in particular, PKCϵ inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

Acute Functional Tolerance to Ethanol Mediated by Protein Kinase Cɛ

A low level of response to ethanol is associated with increased risk of alcoholism. A major determinant of the level of response is the capacity to develop acute functional tolerance (AFT) to ethanol during a single drinking session. Mice lacking protein kinase C epsilon (PKCɛ) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motor-impairing effects of ethanol is reduced in PKCɛ (−/−) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCɛ and resistance of GABAA receptors to ethanol. In contrast, in PKCɛ (−/−) mice, GABAA receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCɛ (−/−) and PKCɛ (+/+) mice developed robust chronic tolerance to ethanol, but the presence of chronic tolerance did not change ethanol preference drinking. These findings suggest that ethanol activates a PKCɛ signaling pathway that contributes to GABAA receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCɛ and does not alter PKCɛ modulation of ethanol preference.

GABAA Receptor Trafficking Is Regulated by Protein Kinase Cε and the N-Ethylmaleimide-Sensitive Factor

Disturbances in GABAA receptor trafficking contribute to several neurological and psychiatric disorders by altering inhibitory neurotransmission. Identifying mechanisms that regulate GABAA receptor trafficking could lead to better understanding of disease pathogenesis and treatment. Here, we show that protein kinase Cε (PKCε) regulates the N-ethylmaleimide-sensitive factor (NSF), an ATPase critical for membrane fusion events, and thereby promotes the trafficking of GABAA receptors. Activation of PKCε decreased cell surface expression of GABAA receptors and attenuated GABAA currents. Activated PKCε associated with NSF, phosphorylated NSF at serine 460 and threonine 461, and increased NSF ATPase activity, which was required for GABAA receptor downregulation. These findings identify new roles for NSF and PKCε in regulating synaptic inhibition through downregulation of GABAA receptors. Reducing NSF activity by inhibiting PKCε could help restore synaptic inhibition in disease states in which it is impaired.

Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior.

Corticotropin‐releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCɛ) null mutant mice, which show reduced anxiety‐like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCɛ activator, ψɛRACK, increased levels of pro‐CRF, whereas reducing PKCɛ levels through RNA interference blocked phorbol ester‐stimulated increases in CRF. Local knockdown of amygdala PKCɛ by RNA interference reduced anxiety‐like behavior in wild‐type mice. Furthermore, local infusion of CRF into the amygdala of PKCɛ−/− mice increased their anxiety‐like behavior. These results are consistent with a novel mechanism of PKCɛ control over anxiety‐like behavior through regulation of CRF in the amygdala.

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

Heterologous Expression of Limulus Rhodopsin

Invertebrates such as Drosophila or Limulus assemble their visual pigment into the specialized rhabdomeric membranes of photoreceptors where phototransduction occurs. We have investigated the biosynthesis of rhodopsin from the Limulus lateral eye with three cell culture expression systems: mammalian COS1 cells, insect Sf9 cells, and amphibian Xenopus oocytes. We extracted and affinity-purified epitope-tagged Limulus rhodopsin expressed from a cDNA or cRNA from these systems. We found that all three culture systems could efficiently synthesize the opsin polypeptide in quantities comparable with that found for bovine opsin. However, none of the systems expressed a protein that stably bound 11-cis-retinal. The protein expressed in COS1 and Sf9 cells appeared to be misfolded, improperly localized, and proteolytically degraded. Similarly, Xenopus oocytes injected with Limulus opsin cRNA did not evoke light-sensitive currents after incubation with 11-cis-retinal. However, injecting Xenopus oocytes with mRNA from Limulus lateral eyes yielded light-dependent conductance changes after incubation with 11-cis-retinal. Also, expressing Limulus opsin cDNA in the R1-R6 photoreceptors of transgenic Drosophila yielded a visual pigment that bound retinal, had normal spectral properties, and coupled to the endogenous phototransduction cascade. These results indicate that Limulus opsin may require one or more photoreceptor-specific proteins for correct folding and/or chromophore binding. This may be a general property of invertebrate opsins and may underlie some of the functional differences between invertebrate and vertebrate visual pigments.

Rhodopsin 5– and Rhodopsin 6–Mediated Clock Synchronization in Drosophila melanogaster Is Independent of Retinal Phospholipase C-β Signaling

Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpAP41) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpAP41 is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.