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It is well established that PI-4,5-P2 generated by type I PIP5K at the plasma membrane is a critical regulator of constitutive endocytosis (Padrón, David, et al. 2003; Nakano‐
Kobayashi, Akiko, et al. 2007; Van den Bout, Iman, 2009). In particular, recruitment and functionality of clathrin adaptor complex AP2 depends on the interaction of AP2 subunits with PIP5K and PI-4,5-P2 (Padrón, David, et al., 2003). PIP5KIα appears to be the major PIP5KI family member providing PI-4,5-P2 during clathrin-mediated endocytosis (Antonescu et al., 2011), whereas PIP5KIγ seems to contribute to clathrin-mediated endocytosis in specific cell types only. Indeed, previous studies have shown that specific ablation of the PIP5KIγ90 isoform in non-neuronal cell types, including murine fibroblasts, does not interfere with clathrin-mediated endocytosis of different plasma membrane receptors (Legate et al., 2011). These findings suggest that PIP5KIα and the remaining PIP5KIγ isoforms are sufficient to allow regular levels of endocytosis in PIP5KIγ90-/- cells and point to other functions of PIP5KIγ90, which might compromise integrin-mediated uptake of S.aureus.
At focal adhesion sites, several integrin- and actin-associated proteins have PI- 4,5-P2
binding capability and assist to connect integrins to the actin cytoskeleton. More precisely, the integrin-associated proteins talin, kindlin, vinculin, FAK as well as α-actinin belong to the core focal adhesion proteins, which are responsive to increased PI-4,5-P2 levels (Janmey, 1994; Toker, A, 2002). However, the source of locally increased PI-4,5-P2 is not completely understood, one possibility is through the recruited PIP5KIγ90 in focal adhesions. In the C-terminus of PIP5KIγ90, there is a short extension (26aa in mouse or 28aa in human) which has the ability to bind to talin.
Via this interaction, PIP5KIγ90 is recruited to the focal adhesions (Legate, Kyle R., et al. 2011). In this regard, it has been demonstrated that recruitment of talin and vinculin, but not kindlin, is affected in PIP5KIγ90-deficient cells (Legate, Kyle R., et al. 2011).
As vinculin is not involved in the uptake of S.aureus (Borisova et al., 2013), a reduced integrin-association of talin in the absence of PIP5KIγ90 might contribute to a diminished internalization of these bacteria. Furthermore, PI-4,5-P2 binding to the talin head domain is required to release intramolecular inhibitory constraints from this ~180 kDa protein and to allow proper orientation of talin at the inner leaflet of the plasma
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membrane (Goksoy et al., 2008; Elliott et al., 2010; Goult et al., 2013). As a consequence, the recruitment and the spatial orientation of talin at integrin-rich focal adhesion sites are impaired in PIP5KIγ90-/- cells, resulting in slower incorporation of talin into new focal adhesion sites (Legate et al., 2011). Therefore, PI-4,5-P2 local production by PIP5KIγ90 could be critical to mobilize talin to newly formed integrin clusters beneath fibronectin-bound bacteria.
In the case of Bartonella henselae, a Gram-negative, facultative intracellular bacterial pathogen, integrins and integrin-associated proteins including talin and FAK are required during host cell invasion (Truttmann et al., 2011). B. henselae binds endothelial cells and injects a panel of bacterial effector proteins into them to induce a peculiar F-actin-based structure, the so-called invasome (Dehio et al., 1997). While B.
henselae does not seem to bind integrins, integrin β1 is nevertheless essential for invasome formation, which in turn mediates the engulfment of the bacteria.
Interestingly, in this context, talin is important for integrin activation via inside-out-signaling, which is a pre-requisite to allow B. henselae induced formation of invasomes (Truttmann et al., 2011). This is in line with the known function of talin during integrin activation, as the talin head domain associates with an NPxY motif in the cytoplasmic tail of integrin subunits (Calderwood et al., 1999; Tadokoro et al., 2003). Talin binding separates the intracellular domains of the integrin α and β subunits, thereby triggering the active, extended conformation of the extracellular domains, which allows ligand binding (Shattil et al., 2010). Though talin might be involved in integrin inside-out signaling during B. henselae uptake, integrin activation in response to cell-matrix adhesion does not seem to be compromised in PIP5KIγ90-deficient cells (Legate et al., 2011). Furthermore, we do not observe an altered binding of fibronectin coated S.aureus to PIP5KIγ90-/- murine fibroblasts suggesting that talin-mediated inside out activation of integrins is not critical for this process or that such a process does not depend on PI-4,5-P2 generated by the PIP5KIγ90 isoform.
As mentioned above, in PIP5KIγ90-/- cells, S.aureus uptake was markedly impaired, talin enrichment is impaired and the re-expression of kinase dead mutant in PIP5Kγ90
-/- cells is not able to rescue the inhibited S.aureus uptake. Together, these results
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demonstrate that the kinase activity and the C-terminal 26aa extension of PIP5Kγ90 are critical for its proper function in S.aureus infection process. However, how the kinase activity of PIP5Kγ90 is regulated during S.aureus uptake is still not known.
There are various researches carried out to investigate how the kinase activity of PIP5KIγ90 and the binding affinity to talin are regulated (Ling, Kun, et al. 2003; Lee, Sang Yoon, et al. 2005; Le, Oanh Thi Tu, et al. 2015). Interestingly, c-Src associates with PIPKIγ90 and the binding affinity is enhanced by FAK. After association, c-Src phosphorylates PIPKIγ90 at the tyrosine 644 (located in the 26aa extension). This tyrosine phosphorylation subsequently enhances the PIPKIγ90-talin interaction and displace β-integrin from talin (Fig.3.3) (Ling, Kun, et al. 2003). Lately, an interesting eight amino acid stretch (from 548 to 555 within protein sequence), R-Y-R-R-R-T-Q-S, was discovered in the C-terminus but outside of the 28 aa extension of PIP5Kγ90.
This sequence matches the Akt consensus phosphorylation motif R-X-R-X-X-T/S (X is any amino acid). Further experiments found that Akt specifically phosphorylates the serine 555 (S555) of PIP5KIγ90 in vitro and in epidermal growth factor (EGF)-treated cells. This serine phosphorylation suppresses PIP5Kγ90–talin interaction and decreases PI-4,5-P2 level. In line with this, the phosphomimetic mutant (S555D), but not the non-phosphorylatable mutant (S555A), of PIP5Kγ90 has a reduced talin binding affinity, lowers PI-4,5-P2 levels, and is dislocated from focal adhesions, as compared to the wild type PIP5Kγ90 (Le, Oanh Thi Tu, et al. 2015). Collectively, these results provided clues about the regulation of PIP5Kγ90 kinase activity in focal adhesion like complexes as well as about the impact of activated PIP5Kγ90 on the integrin-mediated focal adhesion like complexes.
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Fig.3.3 Signaling mechanism depicting PIPKIγ661 (PIPKIγ90) regulation of FAs and the interplay with β-integrin.FAK activates Src and stimulates the association between Src and PIPKIγ661, then, the activated Src phosphorylates PIPKIγ661.Tyrosine phosphorylation of PIPKIγ661 dramatically enhances its interaction with talin and FA targeting, which enable the local generation of PI-4,5-P2 at FAs to improve the integrin–talin interaction. The enhancement of the integrin–talin association by PI-4,5-P2
may ultimately displace PIPKIγ661 from talin, resulting in a reduction of PI-4,5-P2. (Ling, Kun, et al.
2003).