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

Abstract

Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase expressed in many cell types and enriched in neurons. PYK2 is a cytoplasmic enzyme activated by increases in cytosolic free Ca2+ through an unknown mechanism. We report that depolarization or electrical stimulation of hippocampal slices induced a rapid and transient nuclear accumulation of PYK2. Depolarization of cultured neurons or PC12 cells also triggered a Ca2+-dependent nuclear accumulation of PYK2, much more pronounced than that induced by blockade of nuclear export with leptomycin B. Src-family kinase activity, PYK2 autophosphorylation and kinase activity were not required for its nuclear translocation. Depolarization induced a slight decrease in PYK2 apparent molecular mass, compatible with a Ca2+-activated dephosphorylation. Pretreatment of PC12 cells with inhibitors of calcineurin (protein phosphatase 2B), cyclosporin A and FK506, prevented depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2. Transfection with dominant-negative and constitutively active calcineurin-A confirmed the role of calcineurin in the regulation of PYK2 tyrosine phosphorylation and nuclear accumulation. Our results show that depolarization independently induces nuclear translocation and tyrosine phosphorylation of PYK2, and that both responses require calcineurin activation. We suggest that PYK2 exerts some of its actions in the nucleus and that the effects of calcineurin inhibitors may involve PYK2 inhibition.

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

“…Leptomycin B and KN93 were from Calbiochem, cyclosporin A, FK506, forskolin, IBMX, H89, and myristoylated 14-22 fragment of protein kinase A inhibitory peptide (myrPKI [14][15][16][17][18][19][20][21][22] ) from Sigma. Rabbit anti-Pyk2 2-18 antibodies were described [33] or from Sigma, anti-pY402-Pyk2 from Invitrogen, anti-GFP from Roche and anti-DARPP-32 was a gift from P. Greengard (Rockefeller University).…”

“…For fluorescence analyses, PC12 cells were grown in RPMI on type I collagencoated glass coverslips after incubation with poly-L-lysine (Sigma). Depolarization was performed by isosmotic replacement of 40 mM NaCl by 40 mM KCl in the extracellular medium as described previously [20].…”

“…Plasmids encoding the various truncations and point mutations were prepared from full-length GFP-Pyk2 (rat sequence) [20] by site-directed mutagenesis (QuikChange, Stratagene). Briefly, the KpnI fragment of Pyk2 was subcloned into the KpnI site of pBlueScript KS (Fermentas).…”

“…This distribution combined with different levels of expression in various cells precluded the use of automatic detection of nuclear localization. To circumvent this difficulty, we have previously shown that it is possible to estimate the cytonuclear distribution of Pyk2 by quantifying the number of cells in which the nuclear fluorescence is superior or equal to cytoplasmic fluorescence (n C c), with results similar to those obtained by confocal microscopy [20]. Thus, cells were classified by an observer blind to the treatment and/or mutation, using micrographs obtained by epifluorescence microscopy.…”

“…We have previously demonstrated that Pyk2 activation in neuronal cells is concomitant to its Ca 2? -induced, calcineurindependent nuclear accumulation following membrane depolarization [20]. Although Pyk2 nuclear accumulation has been serendipitously observed in various cell types or following mutations [21][22][23][24][25][26][27], the physiological relevance and mechanisms of its cytonuclear shuttling are not known.…”