Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1

Abstract

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

Integrin-linked Kinase Interactions with ELMO2 Modulate Cell Polarity

“…We used laminin 332 as it is the major extracellular matrix protein to which keratinocytes attach in normal, undamaged epidermis (Nguyen et al, 2000). Under these conditions, and in agreement with previous reports (Nikolopoulos and Turner, 2002;Vespa et al, 2003;Nakrieko et al, 2008a), mCherry-ILK was detected throughout the cytoplasm and, at low levels, in the nucleus when expressed in the absence of exogenous ELMO2 ( Figure 5A). GFP-ELMO2 also distributed throughout the cell and, in cells with lamellipodia, ribbons of GFP-ELMO2 fluorescence were visualized in discrete regions of such extensions ( Figure 5A).…”

“…FLAG peptide and all other chemicals were from Sigma. The vectors encoding V5-tagged wild-type and mutant ILK, as well as thioredoxin (TRX)-ILK have been described (Vespa et al, 2005;Nakrieko et al, 2008a). mCherry-tagged ILK was generated by PCR amplification of a V5-tagged human ILK cDNA and cloning into mCherry-C1 (Clontech, Palo Alto, CA).…”

Integrin-linked kinase at a glance

“…Despite its prominent localisation in different integrin adhesion sites, ILK has also been observed in the nucleus of several cell lines, including COS-1 cells (Chun et al, 2005), MCF-7 cells (Acconcia et al, 2007), HeLa cells and keratinocytes (Nakrieko et al, 2008a) (see poster). The nuclear function of ILK, however, is still not well understood.…”

“…For example, it is not known whether the nuclear import of ILK depends on its N-terminus (Acconcia et al, 2007) or on a C-terminal nuclear localisation signal (Chun et al, 2005). The nuclear export of ILK requires the kinase-like domain (Acconcia et al, 2007;Nakrieko et al, 2008a) and is apparently controlled by the nuclear export factor CRM1, integrin-linked kinase-associated serine and threonine phosphatase 2C (ILKAP) and p21-activated kinase 1 (PAK1) (Acconcia et al, 2007;Nakrieko et al, 2008a). …”

“…The nuclear function of ILK, however, is still not well understood. In keratinocytes, nuclear ILK has been shown to induce DNA synthesis (Nakrieko et al, 2008a) and, in MCF-7 cells, it has been found to control the expression of the connector enhancer of kinase suppressor of Ras3 (CNKSR3) gene (Acconcia et al, 2007). It is known that CNKSR3 regulates the epithelial Na + channel (ENaC) through inhibition of the MAPK kinase MEK1 (Ziera et al, 2009).…”