τ kinases in the rat heat shock model: Possible implications for Alzheimer disease

Abstract

We have previously shown, by using the phosphate-dependent anti-antibodies Tau-1 and PHF-1, that heat shock induces rapid dephosphorylation of followed by hyperphosphorylation in female rats. In this study, we analyzed in forebrain homogenates from female Sprague-Dawley rats the activities of extracellular signal regulated kinase 1͞2 (ERK1͞2), c-Jun NH2-terminal kinase (JNK), glycogen synthase kinase-3␤ (GSK-3␤), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase A (PKA), and Ca 2؉ ͞calmodulin-dependent protein kinase II (CaMKII) at 0 (n ‫؍‬ 5), 3 (n ‫؍‬ 4), 6 (n ‫؍‬ 5), and 12 (n ‫؍‬ 5) h after heat shock and in non-heat-shocked controls (n ‫؍‬ 5). Immunoprecipitation kinase assays at 0 h showed suppression of the activities of all kinases except of GSK-3␤, which showed increased activity. At 3-6 h, the activities of ERK1͞2, JNK, Cdk5, and GSK-3␤ toward selective substrates were increased; however, only JNK, Cdk5, and GSK-3␤ but not ERK1͞2 were overactivated toward purified bovine . At 3-6 h, kinase assays specific for PKA and CaMKII showed no increased activity toward either or selective substrates. All of eight anti-antibodies tested showed dephosphorylation at 0 h and hyperphosphorylation at 3-6 h, except for 12E8, which showed hyperphosphorylation also at 0 h. Immunoblot analysis using activity-dependent antibodies against ERK1͞2, JNK, and GSK-3␤ confirmed the above data. Increased activation and inhibition of kinases after heat shock were statistically significant in comparison with controls. Because is hyperphosphorylated in Alzheimer disease these findings suggest that JNK, GSK-3␤, and Cdk5 may play a role in its pathogenesis.

Testosterone prevents the heat shock-induced overactivation of glycogen synthase kinase-3β but not of cyclin-dependent kinase 5 and c-Jun NH 2 -terminal kinase and concomitantly abolishes hyperphosphorylation of τ: Implications for Alzheimer's disease

“…Purification of bovine has been described (19). After excision with Ndel and EcoRI and blunting with the Klenow fragment of E. coli polymerase, the sequence of htau40 containing the entire coding sequence of the longest adult isoform of cerebral was cloned into the Smal cut pQE-32 bacterial expression vector (Qiagen, Chatsworth, CA).…”

“…Protein extracts were prepared exactly as described (19). For GSK-3␤ assay, 100 g of protein extract was precleared with 20 l of protein A-agarose beads for 1 h at 4°C.…”

“…We used phosphospecific Abs to probe inactivated pSer 9 -GSK-3␤ (Cell Signaling Technology, Beverly, MA) and activity-enhanced pTyr 216 -GSK-3␤ (BioSource International, Camarillo, CA) in immunoblots of SDS͞10% PAGE from forebrain extracts prepared as described (19). Incubations in primary Abs were done overnight at 4°C and in goat anti-rabbit IgG coupled to horseradish peroxidase for 1 h at room temperature.…”

“…Fifty micrograms of protein in extracts, prepared as described (19), from control and heat-shocked rats were mixed with an equal volume of 2ϫ Laemmli buffer, underwent 5-12.5% linear gradient PAGE, and were immunostained as above. We used the following three anti-mAbs to correlate the phosphorylation state of with the kinase activities: Tau-1 and PHF-1, which require nonphosphorylated Ser (195͞198͞199͞ 202) and phosphorylated Ser (396͞404), respectively, and the phosphate-independent Tau-5.…”

“…To test this hypothesis, we have shown that heat shock induces hyperphosphorylation of similar to paired helical filament-in female rats (8,16), which is prevented by androgens but not estrogens (17,18). Furthermore, in a recent study, we analyzed six kinases and found that only glycogen synthase kinase-3␤ (GSK-3␤), cyclin-dependent kinase 5 (Cdk5), and c-Jun NH 2 -terminal kinase (JNK) hyperphosphorylated after heat shock (19). We now show that testosterone, but not 17␤-estradiol, prevents hyperphosphorylation of by inhibiting the overactivation of GSK-3␤.…”