Cerebral Ischemia and Seizures Induce Tyrosine Phosphorylation of PYK2 in Neurons and Microglial Cells

Abstract

The nonreceptor tyrosine kinase PYK2 represents a stresssensitive mediator of c-Jun N-terminal kinase and p38 mitogenactivated protein kinase (MAPK) signaling pathways in many cell types. In the present study, we assessed the tyrosine phosphorylation of PYK2 under normal and pathological conditions in the CNS. We generated a polyclonal antibody that selectively recognizes tyrosine-phosphorylated PYK2 at its major autophosphorylation site. By using this antibody, we demonstrate that the phosphorylation profile of PYK2 after focal cerebral ischemia is biphasic. The first phase occurs within 1 hr, when most of the phospho-PYK2 immunoreactivity was observed in cortical neurons, whereas 24-72 hr after ischemia, a striking induction of phospho-PYK2 immunoreactivity was evident in microglia around the necrotic infarcted area. Double-immunostaining analysis using both anti-phospho-PYK2 antibody and antibody against the double-phosphorylated active form of p38MAPK revealed that the two phosphorylated protein kinases exhibit strikingly similar distribution patterns after ischemia. A short time after ischemia, phosphorylation of p38MAPK was evident in the cortical neurons as demonstrated by both immunohistochemistry and immunoblotting analysis, whereas 24-72 hr after ischemia, phospho-p38MAPK was found in activated microglia and colocalized with phospho-PYK2. In contrast to cortical neurons, basal phospho-PYK2 immunoreactivity was observed in hippocampal pyramidal neurons, which was markedly decreased after kainate acid-induced status epilepticus. However, 24 hr after the epileptic onset, a pronounced upregulation of PYK2 and phospho-PYK2 immunoreactivities was evident in microglial cells, as demonstrated by double-immunostaining with the microglial marker OX42. These results provide, for the first time, in situ localization of tyrosine-phosphorylated PYK2 in neuronal stress pathways in the adult rat brain and are consistent with the role of PYK2 as an upstream regulator of p38MAPK signaling cascades in response to stress signals.

Calcineurin is essential for depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2 in neurons

“…Although the non-receptor tyrosine kinase PYK2 was initially described as a cytoplasmic protein, activated by increases in cytosolic free Ca 2+ (Avraham et al, 1995;Sasaki et al, 1995;Yu et al, 1996), in some cells, PYK2 immmunoreactivity is nuclear (Arcucci et al, 2006;Schindler et al, 2007;Tian et al, 2000). The significance of these observations was not clear and our results provide the first evidence for a physiological translocation of PYK2 from the cytoplasm to the nucleus since we show that depolarization or electrical stimulation induced a nuclear accumulation of PYK2 in hippocampal slices.…”

“…By contrast, deleted or mutated forms of PYK2 accumulate in the nucleus of transfected COS-7 cells (Aoto et al, 2002) and PYK2 is localized in the nucleus in chondrocytes and keratinocytes (Arcucci et al, 2006;Schindler et al, 2007). In rat brain, following ischemia or convulsions, PYK2 immunoreactivity appears to be in part nuclear (Tian et al, 2000). However, the significance of these observations is not known.…”

“…PYK2 is phosphorylated on tyrosine after depolarization (Siciliano et al, 1996) and its activation appears to be necessary for the induction of LTP (Huang et al, 2001). In addition, PYK2 is activated in the hippocampus following brain ischemia and convulsions (Tian et al, 2000) suggesting a role in these pathologies. PYK2 is also involved in many other biological functions including activation of macrophages (Okigaki et al, 2003) and osteoclasts (Lakkakorpi et al, 2003), as well as in pathological conditions including cancer (Lipinski et al, 2003) and cardiac hypertrophy (Hirotani et al, 2004).…”

Microglial Activation in Stroke: Therapeutic Targets

“…p38 MAPK promotes the stabilization and enhanced translation of mRNAs encoding proinflammatory proteins. 147 Activated (phosphorylated) p38 has been demonstrated in microglia in animal models of brain ischemia, 146,148,149 and pharmacological inhibition of p38 with the compound SD-282 decreased the number of activated microglia in ischemic brain. 150 The MAPK/ ERK signaling pathway may also regulate inflammation through its effects on PARP-1 activation, which (as detailed later in this review) is an important modulator of proinflammatory gene expression.…”

Extracellular Signal-Regulated Kinase 1/2 Is Required for the Induction of Group I Metabotropic Glutamate Receptor-Mediated Epileptiform Discharges

“…Previous studies have shown that epileptiform activities in various experimental models are associated with alterations in the level of MAPK phosphorylation (Garrido et al, 1998;Mielke et al, 1999;Tian et al, 2000). In addition, it has been shown that inhibition of the ERK1/2 pathway attenuates the frequency and duration of epileptiform discharges (Murray et al, 1998;Sanna et al, 2000).…”