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 ␣12␥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...
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