Te Molder Lisa, Sonnenberg Arnoud
The Division of Cell Biology, The Netherlands Cancer Inst., Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
PLoS One. 2015 Nov 18;10(11):e0143357. doi: 10.1371/journal.pone.0143357. eCollection 2015.
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.
整合素α6β4是半桥粒（HDs）的主要成分，通过与细胞骨架连接蛋白网蛋白结合并与角蛋白丝相关联，稳定角质形成细胞与表皮基底膜的细胞粘附。β4亚基磷酸化导致α6β4 - 网蛋白相互作用破坏，从而使角质形成细胞与层粘连蛋白-332的粘附强度降低，半桥粒溶解。此前，我们已证明β4胞质结构域C末端的T1736磷酸化会破坏β4与网蛋白的 plak结构域的相互作用。此外，我们表明β4 - T1736在体外可被PKD1磷酸化，尽管PMA和EGF均诱导T1736磷酸化，但只有PMA能够激活PKD1。在此，我们表明细胞内钙离子浓度（[Ca2+]i）的降低增强了PMA和EGF诱导的β4 - T1736磷酸化，这是由于钙敏感蛋白磷酸酶钙调神经磷酸酶的抑制和ERK1/2激活的增强所致。我们还表明，在角质形成细胞中，PMA刺激的β4 - T1736磷酸化主要由PKCδ下游的PKD2激活介导。另一方面，EGF刺激的T1736磷酸化和EGF诱导的半桥粒溶解均依赖于功能性MAPK信号通路，用RSK抑制剂BI - D1870处理可阻止EGF刺激的β4 - T1736磷酸化。此外，野生型RSK1的过表达增强了β4 - T1736的磷酸化，而激酶失活的RSK1的表达或通过siRNA介导的RSK1缺失则使其降低。总之，我们的数据表明不同的刺激可导致PKD2或RSK1对β4 - T1736的磷酸化。
