The Role of Proline-Rich Protein Tyrosine Kinase 2 in Differentiation-Dependent Signaling in Human Epidermal Keratinocytes

Abstract

Non-receptor tyrosine kinase proline-rich protein tyrosine kinase 2 (Pyk2) functions as an integrator of multiple signaling pathways involved in the regulation of fundamental cellular processes. Pyk2 expression, regulation, and functions in skin have not been examined. Here we investigated the expression and subcellular localization of Pyk2 in human epidermis and in primary human keratinocytes, and studied the mechanisms of Pyk2 activation by differentiation-inducing stimuli, and the role of Pyk2 as a regulator of keratinocyte differentiation. We demonstrate that Pyk2 is abundantly expressed in skin keratinocytes. Notably, the endogenous Pyk2 protein is predominantly localized in keratinocyte nuclei throughout all layers of healthy human epidermis, and in cultured human keratinocytes. Pyk2 is activated by treatment with keratinocyte-differentiating agents, 12-O-tetradecanoylphorbol-13-acetate and calcium via a mechanism that requires intracellular calcium release and functional protein kinase C (PKC) and Src activities. Particularly, differentiation-promoting PKC delta and PKC eta elicit Pyk2 activation. Our data show that Pyk2 increases promoter activity and endogenous protein levels of involucrin, a marker of keratinocyte terminal differentiation. This regulation is associated with increased expression of Fra-1 and JunD, activator protein-1 transcription factors known to be required for involucrin expression. Altogether, these results provide insights into Pyk2 signaling in epidermis and reveal a novel role for Pyk2 in regulation of keratinocyte differentiation.

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.…”

“…Although it is likely that PYK2 plays an important role in post-synaptic densities, because of its interaction with NMDA receptors and associated scaffold proteins (Bongiorno-Borbone et al, 2005;Cheung et al, 2000;Heidinger et al, 2002;Liu et al, 2001;Seabold et al, 2003), it does not appear to be enriched in spines. 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).…”

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

“…Interesting clues are available from various models. Nuclear Pyk2 activates keratinocytes differentiation by increasing the expression of Fra-1 and JunD transcription factors [24]. The autophosphorylated, active form of Pyk2 accumulates in the nucleus of depolarized neurons [20], although Pyk2 nuclear accumulation is independent of its autophosphorylation and kinase activity ( [20] and present study).…”

“…-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. Cytonuclear trafficking of proteins larger than 40 kDa results from an active transport mediated by karyopherins, which facilitate translocation of cargos through the nuclear pore and release them in the nucleus, in the case of importins, or in the cytoplasm in the case of exportins [28,29].…”

Effect of protein kinase C and Ca2+ on p42/p44 MAPK, Pyk2, and Src activation in rat conjunctival goblet cells

“…Similar to conjunctival goblet cells, activation of Pyk2 by PKC and [Ca 2+ ] i has been reported in PC12 cells (Lev, et al, 1995) and other tissues as well , Schindler, et al, 2007, Wang and Reiser, 2003. Phosphorylation of Src on Tyr 416 has been shown up-regulate the activity of Src (Xu, et al, 1999) while Pyk2, upon activation, undergoes autophosphorylation on Tyr 402 (Dikic, et al, 1996, Park, et al, 2004.…”