Serine Phosphorylation of the Integrin β4 Subunit Is Necessary for Epidermal Growth Factor Receptor–induced Hemidesmosome Disruption

Abstract

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of ␣6␤4 to plectin plays a central role in their assembly. We have defined three regions on ␤4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of ␤4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of ␤4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the ␤4 subunit.

The making of hemidesmosome structures in vivo

“…For example, the phosphorylation state of the b4-integrin cytoplasmic domain determines the strength of its interaction with plectin, and may act as a regulation point for hemidesmosome assembly and disassembly. Several protein kinases can phosphorylate b4-integrin, including PKC, PKA, the Epidermal Growth Factor Receptor, and the Macrophage Stimulating Factor Receptor (Santoro et al, 2003;Rabinovitz et al, 2004;Wilhelmsen et al, 2007). However, controversy remains in explaining their roles in hemidesmosome formation due to unphysiological experimental conditions (Margadant et al, 2008).…”

The Calcium/Calcineurin Pathway Promotes Hemidesmosome Stability through Inhibition of β4 Integrin Phosphorylation

“…There is little information about serine-threonine protein phosphatases (PPP) that might be involved in this process. PPP inhibitors, such as okadaic acid and calyculin, have been shown to increase ␤4 phosphorylation as well as destabilize HD, implicating protein phosphatase 2a and or protein phosphatase 1 (18,19). In the present study we addressed whether another major PPP, calcineurin (CN), is involved in the dephosphorylation and function of the ␤4 integrin.…”

“…A cluster of serines at the beginning of the connecting segment, Ser 1356 -Ser 1360 -Ser 1364 , as well as at a nearby site, Ser 1424 (17)(18)(19)(20), or a threonine at the C-tail (21), have been shown to play an important role in HD stability. Mutation on some of these serine residues to alanine to impede phosphorylation reduces the disruptive activity of EGF on HD, increases the interaction of ␤4 and plectin, and can inhibit cell migration by stabilizing HD, indicating a function of ␤4 serine phosphorylation in HD regulation (17)(18)(19)(20). In contrast, mutation of these sites into aspartate, which mimics phosphorylation, increases HD disruption and plectin dissociation.…”

“…Although several kinases, such as PKC and the MAPK ERK1/2 and RSK2, have been shown to be involved in serine phosphorylation of ␤4 (16 -20), little is known about the pathways that lead to dephosphorylation. Based on mutational studies, it can be asserted that dephosphorylation of the ␤4 integrin is necessary to stabilize HD (17)(18)(19)(20). There is little information about serine-threonine protein phosphatases (PPP) that might be involved in this process.…”

Curcumin Inhibition of the Functional Interaction between Integrin α6β4 and the Epidermal Growth Factor Receptor

“…This is where a6b4 interacts with growth factor receptors, leading to interaction with the actin cytoskeleton important to cancer cell invasion and metastasis (17,31). Phosphorylation of key serine residues (S1356, 1360, 1364, 1424) of b4 integrin by PKC-a is involved in HD disassembly and the mobilization of a6b4 from HDs (34)(35)(36). Therefore, it is possible that curcumin may affect one of the signaling pathways responsible for the phosphorylation of these Ser residues.…”