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 ATPgammaS 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, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, 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.

Ethanol enhances ␥-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA A receptors, especially at low concentrations achieved during social drinking. We postulated that the ⑀ isoform of protein kinase C (PKC⑀) regulates the ethanol sensitivity of GABA A receptors, as mice lacking PKC⑀ show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKC⑀ mutant to selectively inhibit the catalytic activity of PKC⑀. We used this mutant and PKC⑀ ؊/؊ mice to determine that PKC⑀ phosphorylates ␥2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at ␣1␤2␥2 receptors, which is the most abundant GABA A receptor subtype in the brain. Our findings indicate that PKC⑀ phosphorylation of ␥2 regulates the response of GABA A receptors to specific allosteric modulators, and, in particular, PKC⑀ inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol. ␥-Aminobutyrate type A (GABA A )3 receptors mediate the majority of rapid inhibitory neurotransmission in the brain and are an important target for ethanol, the most widely abused drug (1). Ethanol modulation of GABA A receptors was first identified in synaptosomal preparations where intoxicating concentrations (10 -30 mM) enhanced receptor function as measured by 36 Cl uptake assays (2, 3). However, after 30 years of investigation, it is apparent that ethanol enhancement of synaptic GABA A receptors is variable and in some preparations cannot be detected even at anesthetic concentrations (1, 4).GABA A receptors are pentameric protein complexes of subunits from eight classes (␣1-6, ␤1-3, ␥1-3, ␦, ⑀, , , and 1-3) (5). Most receptors are composed of two ␣ subunits and two ␤ subunits that co-assemble with one ␥2 subunit, which anchors these receptors at synapses where they mediate phasic inhibition (6). A minority contain a ␦ subunit instead of ␥2; these receptors are extrasynaptic and mediate tonic inhibition in neurons (7). Because earlier ethanol studies that focused on GABA currents carried by ␥2-containing receptors produced variable results, recent attention has turned to ethanol effects at receptors containing ␦ subunits. Reports from three laboratories found these receptors enhanced by low (Յ30 mM) intoxicating concentrations of ethanol (8 -10). However, two recent studies were unable to demonstrate low dose ethanol sensitivity of ␦-containing GABA A receptors (11, 12), indicating that, like synaptic GABA A receptors, ethanol modulation of extrasynaptic receptors is also variable.The reasons for this high degree of variability are unknown. One possibility is that intracellular signaling pathways may regulate ethanol sensitivity of GABA A receptors, and the activity of such pathways was not controlled for in these studies. This hypothesis is consistent with our findings in mice lacking protein kinase C⑀ (PKC⑀), which show an increased behavioral response to ethanol (13). Ethanol modulation of GABA A receptors is a...

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. Video LinkThe video component of this article can be found at https://www.jove.com/video/2761/ Protocol MCAO MethodThis protocol was approved by the Institutional Animal Care and Use Committees at UCSF and Kent State University, and abides by the National Institutes of Health guidelines for the use of experimental animals.1. Cut a 5-0 monofilament suture (Harvard Apparatus, Holliston, MA) into 20 mm segments. Round the tip of each segment by heating it near a cauterizer (Braintree Scientific, Inc., Braintree, MA). Measure the diameter of the tip using a micrometer (Applied Image Inc., Rochester, NY). We use a suture with a final tip diameter of 0.21-0.22 mm for a mouse with body weight of 25-30 g. 2. Sterilize all surgical tools by autoclaving (minimum 121 °C, 15 PSI, for 15 min). Sanitize the surgery table and associated equipment using 70% ethanol. 3. Anesthetize an 8-12 week-old mouse (25-30 g) with 5% isoflurane (Aerrane, Baxter, Deerfield, IL) in 30% O 2 / 70% N 2 O using the V-10 Anesthesia system (VetEquip, Inc., Pleasanton, CA). Following induction of anesthesia, reduce the level of isoflurane and maintain it at 1.5%. 4. Place the mouse in the supine position on a heating pad. Insert a rectal probe, and monitor and maintain body temperature between 36.5-37.5 °C using the TR-200 homeothermic temperature system (Fine Science Tools Inc., Foster City, CA). 5. Shave the fur on the ventral neck region with electric clippers (Braintree Scientific) to expose the skin. Disinfect the surgica...

Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin-2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose-dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce 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.

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 (PKCe) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motorimpairing effects of ethanol is reduced in PKCe (À/À) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCe and resistance of GABA A receptors to ethanol. In contrast, in PKCe (À/À) mice, GABA A receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCe (À/À) and PKCe ( + / + ) 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 PKCe signaling pathway that contributes to GABA A receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCe and does not alter PKCe modulation of ethanol preference.

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