Our data suggest a pathway where pIgA binding to pIgR activates Yes, followed by activation of EGFR and then the MEK–ERK module (presumably through Ras and Raf), which culminates in phosphorylation of FIP5, control of Rab11a localization and stimulation of transcytosis (). Earlier work indicated that regulation of pIgA transcytosis also involves calcium, kinases (protein kinase C; PKC and phosphatidylinositol 3-kinase; PI3Kinase), numerous Rabs (Rab3b, Rab5, Rab11a, Rab17 and Rab25), retromer and signals in the pIgR cytoplasmic domain4,5,27–30
. Thus, this cascade is part of a complex network governing transcytosis.
Figure 8 A kinase cascade regulating pIgR transcytosis. (a) A Yes–EGFR–ERK–FIP5 signalling cascade controls pIgA–pIgR transcytosis in epithelial cells. pIgA stimulates pIgR activation, which associates with Yes, and directs phosphorylation (more ...)
pIgR, EGFR and Yes formed a complex in sub-apical endosomes, which were devoid of the ARE marker Rab11a, suggesting that the complex may occur in apically located common recycling endosomes (CREs), before ARE entry (). pIgA stimulated dispersal of the complex, concomitant with apical transcytosis of pIgA–pIgR, which occurs through the ARE under control of Rab11a21,31
. EGFR knockdown not only attenuated pIgA-induced pIgR transcytosis, but also increased transport of non-receptor-bound pIgR to both the apical and basolateral surfaces, suggesting a fundamental requirement for EGFR in regulating pIgR transport to the apical surface.
Rab11a and FIP5 regulate pIgA transcytosis14,31
. Here, we demonstrate that ERK phosphorylation on FIP5 Ser 188 is crucial for efficient pIgA–pIgR transcytosis. pIgA-induced FIP5 phosphorylation is blocked by inhibitors of SFK, EGFR or MEK. Expression of a FIP5S188A
mutant disrupted polarized distribution of pIgA and Rab11a and led to co-accumulation of Rab11a/FIP5-labelled vesicles in the periphery of the sub-apical region, suggesting that Ser 188 phosphorylation controls localization of FIP5 and Rab11a. FIP5S188A
also functioned as a dominant-negative inhibitor of pIgA-induced transcytosis, suggesting that this EGFR/MEK/ERK target is a critical residue in the regulation of transcytosis.
EGFR activation by tyrosine phosphorylation may regulate this network by coupling EGFR to numerous effectors with SH2 domains32–34
. For example, EGFR phosphorylated at Tyr 992 phosphorylates and activates PLCγ35,36
. Phosphorylation of PLCγ1 increases in MDCK cells on pIgA binding24
. The resultant phospholipid hydrolysis leads to activation of PKC and elevation of intracellular free calcium; both promote pIgR transcytosis37,38
. We now show that pIgA binding leads to phosphorylation of EGFR mainly at Tyr 992, Tyr 1173 and Tyr 845. The role of ERKs in pIgA transcytosis was focused on because ERK can be activated by phosphorylation of EGFR Tyr 1173/Tyr 992 through the Ras–Raf–MEK pathway39–41
. ERK is abundant in rat liver endosomes and we show that EGFR functions, at least partly, by coupling pIgR to ERK, which is activated rapidly after pIgA stimulation. pIgA-induced phosphorylation of ERK was blocked by depleting or inhibiting either Yes or EGFR, suggesting that EGFR activates the MEK/ERK pathway in pIgR-containing endosomes.
This pathway links kinases that are traditionally viewed as regulating development (EGFR–MAPK–ERK)42
with regulators of membrane traffic (Rab11a and FIP5). An explanation for this unusual connection is that the levels of phosphorylation and activation of these signalling components by pIgA are lower than usually seen in regulation of development (though statistically significant and reproducible). Indeed, constitutive EGFR activation perturbed, rather than promoted, transcytosis. Notably, ERK has also been found to directly phosphorylate and control the function of protrudin, another Rab11-interacting protein that regulates polarized endocytic sorting43
. Furthermore, inhibition of the MEK–ERK pathway perturbs endosome morphology and recycling of molecules in ARF6-positive recycling vesicles40
. Thus, in addition to the bistable regulation of developmental processes often associated with EGFR and kinase cascades, our data support an emerging model that endocytic machinery may be a common, but unappreciated, target of ERKs regulating membrane traffic in diverse contexts. Regulation of pIgA transcytosis involves transmission of information across the cell; ERK signalling is suited for such long distance signal transmission44
Regulation of membrane traffic is a central issue in cell biology. Indeed, many types of physiological adaptation as well as most developmental events involve regulation of membrane traffic45
. One general type of traffic regulation is that the level of cargo can regulate its own transport. For example, an increase in the amount of newly synthesized secretory protein in the endoplasmic reticulum can lead to an increase in the amount of chaperones needed to properly fold the cargo, as well as in the amount of vesicular traffic leaving the endoplasmic reticulum46
. The ability of the pIgR to increase its transcytosis in response to an increase in the amount of pIgA is a good model of this type of autoregulation.
Autoregulation of pIgA transcytosis is probably medically important. In response to mucosal infection, pIgA production can rapidly increase and autoregulation provides for its efficient transport into secretions. pIgA often forms a complex with antigen47
and failure to adequately transport such antigen–antibody complexes may lead to their pathological deposition, such as in IgA nephropathy, a major cause of kidney failure worldwide48
. Moreover, in IgA nephropathy, IgA complexes are abnormally deposited in renal glomeruli. This might cause abnormal activation of signalling by EGFR (or members of the EGFR family) leading to pathological proliferation, a hallmark of IgA nephropathy. The regulation of transcytosis by EGFR provides a rapid, post-transcriptional mechanism for coordinating response to infection or injury with mucosal immunity. pIgR is also transcriptionally upregulated by several cytokines, providing a complementary mechanism to coordinate pIgA transcytosis with mucosal immunity49