Phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 may not always represent its kinase activity in a rat model of focal cerebral ischemia with or without ischemic preconditioning

Abstract

The extracellular signal-regulated kinase (ERK) 1/2 protein requires a dual phosphorylation at conserved threonine and tyrosine residues to be fully activated under normal physiological conditions. Thus, ERK1/2 kinase activity is often defined by the quantity of phosphorylated kinase. However, this may not accurately represent its true activity under certain pathological conditions. We investigated whether ERK1/2 kinase activity is proportional to its phosphorylation state in a rat focal ischemia model with and without rapid ischemic preconditioning. We showed that phosphorylated-ERK1/2 protein levels were increased 2.6±0.07-fold, and ERK1/2 kinase activity was increased 10.6±1.9-fold in animals receiving ischemic preconditioning alone without test ischemia compared with sham group (P<0.05, n=6/group), suggesting that phosphorylated-ERK1/2 protein levels represent its kinase activity under these conditions. However, preconditioning plus test ischemia robustly blocked ERK1/2 kinase activity, whereas it increased phosphorylated-ERK1/2 protein levels beyond those receiving test ischemia alone, suggesting that phosphorylated-ERK1/2 protein levels were not representative of actual kinase activity in this pathological condition. In conclusion, protein phosphorylation levels of ERK1/2 do not always correspond to kinase activity, thus, measuring the true kinase activity is essential.

Fig. 1. Rapid preconditioning inhibited infarct size

A. Representative infarction with TTC staining. Ischemic brains were harvested 2 d after stroke for TTC staining, sectioned into 5 blocks, and stained with TTC solution. B. Bar graphs (the right column) representing the average infarction size normalized to the non-ischemic cortex. N=7/group. ** vs control ischemia (30 min), P<0.01. C. Diagram showing the ischemic tissues corresponding to ischemic penumbra and core that were dissected for analysis. Ischemic penumbra (region I) is defined as the ischemic region spared by preconditioning; the ischemic core (region II) refers to the ischemic area that develops into infarction in animals receiving preconditioning and test ischemia. In the test ischemic group, a line defining the ischemic border was drawn 1.5 mm lateral to the midline. From this line, an approximately 2mm wide area of ischemic tissue was dissected as region I. The remaining ischemic tissue after dissection of region I was dissected as the ischemic core. The corresponding regions in the preconditioning animals were dissected as ischemic penumbra and core.

Fig. 2. ERK1/2 phosphorylation and activity was increased in rats receiving ischemic preconditioning alone without stroke

Ischemic preconditioning was performed 1 h prior to permanent MCAo by transient MCAo with a micro clip for 15 min. Rat brains were harvested 1 h after preconditioning; no test ischemia was induced. The brain region corresponding to ischemic penumbra (region I defined in Fig.1) was dissected for analysis. Rat brains were processed for Western blot and ERK1/2 kinase assay. A. Western blot showing RIPC increased p-ERK1/2 levels (top). Two protein bands were observed. The top band is pERK1 (44 kDa), and the lower band is pERK2 (42kDa). The bar graph shows the average optical densities of the protein bands (bottom). B. RIPC also increased ERK1/2 activity as assessed by in vitro kinase assay compared to sham. Activated protein kinase phosphorylates its substrates, thus levels of a phophorylated protein substrate can be used as a marker for protein kinase activity. In this experiment, ERK kinase activity was determined by the amount of Elk-1 phosphorylated by ERK1/2; p-Elk-1 was detected by Western blot. * vs sham, P<0.05. N=3–6/group.

Fig. 3. The effects of stroke and ischemic preconditioning on p-ERK1/2 and ERK1/2 activity

A. Representative protein bands of p-ERK1/2 and ERK1/2 in the ischemic core 1, 5 and 24 h after stroke. The bar graphs show the mean value of p-Erk1/2 optical densities. No significant changes in ERK1/2 protein bands were detected after stroke, and RIPC did not affect its expression (statistical results are not shown). β-actin was probed to show even protein loading. pERK1/2 was increased after stroke, and RIPC further increased its levels at 1 h after stroke in the ischemic core. B. Representative protein bands of p-Erk1/2 and ERK1/2 in the ischemic penumbra. The bar graphs represented the mean values of protein densities, which were normalized to the values of the sham group. N=3–6/group *, **, *** vs sham, P<0.05. 0.01. 0.001, respectively. # vs control (test) ischemia, P<0.05.

Fig. 4. The effects of stroke on ERK1/2 kinase activity assessed by in vitro kinase assay

Ischemic tissues corresponding to penumbra were dissected from animals 1, 5 and 24 h after stroke with or without RIPC. Cell lysates were prepared, co-immunoprecipitation with p-ERK1/2 antibodies was performed, and extracted p-ERK proteins were incubated with Elk-1 protein. p-Elk-1 was measured by Western blot. The amount of p-Elk-1 represents ERK1/2 kinase activity, which was significantly increased at 1 and 5 h after stroke, which preconditioning blocked. N=4–6/group. * vs sham, P<0.05; # vs control, P<0.05.