PKD2 and RSK1 Regulate Integrin β4 Phosphorylation at Threonine 1736.

The integrin α6β4, a major component of hemidesmosomes (HDs), stabilizes keratinocyte cell adhesion to the epidermal basement membrane through binding to the cytoskeletal linker protein plectin and association with keratin filaments. Disruption of the α6β4-plectin interaction through phosphorylation of the β4 subunit results in a reduction in adhesive strength of keratinocytes to laminin-332 and the dissolution of HDs. Previously, we have demonstrated that phosphorylation of T1736 in the C-terminal end of the β4 cytoplasmic domain disrupts the interaction of β4 with the plakin domain of plectin. Furthermore, we showed that β4-T1736 can be phosphorylated by PKD1 in vitro, and although both PMA and EGF induced T1736 phosphorylation, only PMA was able to activate PKD1. Here, we show that depletion of [Ca2+]i augments PMA- and EGF-induced phosphorylation of β4-T1736 and that this is caused by inhibition of the calcium-sensitive protein phosphatase calcineurin and augmentation of ERK1/2 activation. We also show that in keratinocytes the PMA-stimulated phosphorylation of β4-T1736 primarily is mediated by PKD2 activation downstream of PKCδ. On the other hand, both the EGF-stimulated phosphorylation of T1736 and the EGF-induced dissolution of HDs are dependent on a functional MAPK signaling pathway, and treatment with the RSK inhibitor BI-D1870 prevented EGF-stimulated phosphorylation of β4-T1736. Moreover, phosphorylation of β4-T1736 is enhanced by overexpression of wild-type RSK1, while it is reduced by the expression of kinase-inactive RSK1 or by siRNA-mediated depletion of RSK1. In summary, our data indicate that different stimuli can lead to the phosphorylation of β4-T1736 by either PKD2 or RSK1

EGF- but not PMA-stimulated phosphorylation of β4 at T1736 and S1364 depends on RSK1/2.

<p>PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM for 30 min and/or BI-D1870 for 30 min at the indicated concentrations or left untreated and then stimulated with EGF or PMA. The cell lysates were analysed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and β4-S1364 proteins after normalization to the level of α-tubulin.</p

Regulation of hemidesmosome formation and β4-T1736 phosphorylation by extracellular calcium.

<p><i>A</i>, Confocal microscopic images show the effect of extracellular calcium on the formation and organization of HDs in PA-JEB/β4 keratinocytes. After 24 hours of growth in either low (KGM; left picture) or high calcium (DMEM; right picture) medium, PA-JEB/β4 keratinocytes were stained with mAbs 439-9B against β4 (green) and 31 against plectin (red). Cells were counterstained with DAPI (bleu) to identify nuclei. HDs are identified as dots containing both β4 and plectin and appear in yellow. Scale bars: 20 μm. <i>B</i>, Co-localization between β4 and plectin was quantified for each cell using Pearson’s correlation coefficient. Box plots show median (solid line) and 25th and 75th percentiles (boxes) of the distribution for each condition (n = 3, ~10 images per experiment). **** <0,0001. <i>C</i>, PA-JEB/β4 keratinocytes were pre-treated with the intracellular calcium chelator, BAPTA-AM and/or different concentrations of the calcium ionophore A23187 (1–5 μM), or left untreated. Cell lysates were analyzed by immunoblotting with antibodies as indicated. <i>D</i>, PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM, different concentrations (5–100 nM) of FK506 or left untreated before stimulation with PMA. Cell lysates were analyzed by immunoblotting with the indicated antibodies. <i>E</i>. PA-JEB/β4 keratinocytes and A431 epidermoid carcinoma cells were serum-starved overnight, pre-treated with BAPTA-AM or different concentrations of FK506 (5–100 nM) or CsA (50–250 nM) and stimulated with PMA or left unstimulated. Cell lysates were analyzed by immunoblotting with the indicated antibodies. <i>F</i>. Schematic diagram of the different stimuli and inhibitors along with the signaling pathways that control β4 phosphorylation. Cn, calcineurin; CsA, Cyclosporin A; FK506, tacrolimus; BAPTA-AM, 1,2-<i>Bis</i>(2-aminophenoxy)ethane-<i>N</i>,<i>N</i>,<i>N</i>',<i>N</i>'-tetraacetic acid tetrakis (acetoxymethyl ester).</p

EGF-induced HD dissolution is dependent on MAPK signaling.

<p>PA-JEB/β4 keratinocytes, seeded on coverslips, were deprived of growth factors overnight, pre-treated with the MEK inhibitor UO126 and/or stimulated with EGF. <i>A</i>. PA-JEB/β4 keratinocytes were stained with mAbs 439-9B against β4 (green) and 31 against plectin (red). Cells were counterstained with DAPI (blue). Co-localization of β4 and plectin in HDs appears in yellow. Scale bars: 20 μm. <i>B</i>. Co-localization between β4 and plectin was quantified using Pearson’s correlation coefficient for each image. Box plots show median (solid line) and 25th and 75th percentiles (boxes) of the distribution for each condition (n = 4, ~20 images per experiment). ****<i>P</i>, <0.0001.</p

PKCδ mediates PMA-induced PKD activation.

<p><i>A</i>. Expression levels of the indicated mRNAs for the four novel PKCs were determined by RT-qPCR and normalized to cyclophilin A mRNA levels in PA-JEB/β4 cells. Values are means ± SD from four independent experiments performed in duplo. <i>B</i>. Relative mRNA levels of PKCδ, PKCε and PKCη were determined by RT-qPCR in PA-JEB/β4 cells, transfected with control (Ctrl) siRNA or siRNAs targeting PKCε, PKCδ, or PKCη. The values were normalized to cyclophilin A mRNA and compared to the mRNA levels in the cells treated with control siRNA. They are the means ± SD from four independent experiments performed in duplo. ****<i>P</i>, <0.0001; ***<i>P</i>, <0.001; **<i>P</i>, <0,01; *<i>P</i>, <0,05. <i>C</i>, <i>D</i>. PA-JEB/β4 keratinocytes were transfected with single siRNAs or combinations of siRNAs as indicated. After 15 h, the cells were deprived of growth factors for 24h and subsequently stimulated with PMA for 10 min or left unstimulated. Cell lysates were analyzed by immunoblotting with antibodies as indicated. <i>E</i>. PA-JEB/β4 keratinocytes, transfected with control, PKCδ or PKCδ siRNA in combination with PKCε and PKCη siRNAs, were deprived of growth factors for 24h, pre-treated with BAPTA-AM for 1h and subsequently treated with PMA for 10 min or left untreated and stimulated. Cell lysates were analyzed by immunoblotting with antibodies as indicated. The values below the blots shown in panels C, D and E indicate the signal intensities for the phosphorylated β4-T1736 and PKD (S744/748) proteins after normalization to the level of α-tubulin.</p

PMA- and EGF-stimulated phosphorylation of β4-T1736 is dependent on PKC and MAPK, respectively.

<p>PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM for 30 min and then treated for 30 min with different concentrations of Gӧ6983 (100 nM-1 μM) or UO126 (2–10 μM). Subsequently the cells were stimulated with EGF or PMA for 10 min. Cell lysates were analyzed by immunoblotting with antibodies as indicated. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and β4-S1364 proteins after normalization to the level of β4.</p

EGF-stimulated phosphorylation of β4 at T1736 is mediated by RSK1.

<p><i>A</i>. PA-JEB/β4 keratinocytes, transiently overexpressing RSK1, RSK2, kinase-dead RSK1 or kinase-dead RSK2, were deprived of growth factors overnight and stimulated with EGF or left unstimulated. The cell lysates were analysed by immunoblotting with antibodies as indicated. <i>B</i>. PA-JEB/β4 keratinocytes transfected with RSK1 and/or RSK2 siRNAs were deprived of growth factors overnight and stimulated with EGF or left unstimulated. The cell lysates were analysed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and β4-S1364 proteins after normalization to the level of α-tubulin.</p

Signaling pathways controling β4 phosphorylation at serine and threonine residues.

<p>Signaling pathways controling β4 phosphorylation at serine and threonine residues.</p

PMA-stimulated phosphorylation of β4-T1736 is primarily mediated by PKD2.

<p><i>A</i>. PA-JEB/β4 keratinocytes, not transfected (-) or transfected with siRNAs against PKD1 or PKD2, or a combination of PKD1 and PKD2 siRNAs, were deprived of growth factors overnight, pre-treated with UO126 (10 μM) or left untreated, and then stimulated with PMA for 10 min. The cell lysates were analyzed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 and PKD (S744/748) proteins after normalization to the level of α-tubulin. <i>B</i>. Expression levels of the mRNAs for the three PKD family members were determined by RT-qPCR relative to cyclophilin A mRNA levels in PA-JEB/β4 cells. Values are means ± SD from four independent experiments performed in duplo.</p

PI3K and mTOR are not required for EGF-mediated β4 phosphorylation.

<p>PA-JEB/β4 keratinocytes were deprived of growth factors overnight, pre-treated with BAPTA-AM and/or different concentrations of the mTOR kinase inhibitor AZD 8055 (0,8–100 nM) or the PI3K inhibitor GDC-0941 (8 nM- 1 μM) and subsequently stimulated with EGF or left unstimulated. The cell lysates were analysed by immunoblotting with the indicated antibodies. The values below the blots indicate the signal intensities for the phosphorylated β4-T1736 after normalization to the level of α-tubulin.</p