PI3Kγ is a novel regulator of TNFα signaling in the human colon cell line HT29/B6.

Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes phosphorylating phospholipids in the membrane, thereby, promoting the PI3K/AKT signaling cascade. PI3Ks are involved in a variety of fundamental cellular functions, including tumor necrosis factor α (TNFα)-induced tight junction (TJ) impairment-a hallmark of inflammatory bowel diseases. Most of the studies analyzing the role of class I PI3K signaling in epithelial barrier maintenance did not decipher which of the isoforms are responsible for the observed effects. By using wild-type and PI3Kγ-deficient HT-29/B6 cells, we characterized the functional role of PI3Kγ in these cells under inflammatory conditions. Measurement of the transepithelial electrical resistance and the paracellular flux of macromolecules revealed that monolayers of PI3Kγ-deficient cells, compared with wild-type cells, were protected against TNFα-induced barrier dysfunction. This effect was independent of any PI3K activity because treatment with a pan-PI3K inhibitor did not alter this observation. By immunostaining, we found correlative changes in the distribution of the TJ marker ZO-1. Furthermore, the absence of PI3Kγ reduced the basal level of the pore-forming TJ protein claudin-2. Our study suggests a novel noncanonical, kinase-independent scaffolding function of PI3Kγ in TNFα-induced barrier dysfunction.

Influence of liposome composition and membrane binding on protein kinase activity of PI3Kγ.

Phosphoinositide 3-kinase γ (PI3Kγ) has been implicated in a variety of cellular signaling processes. It is a multifunctional enzyme with lipid and protein kinase activity that also acts as a scaffold protein. Although it is well known that membrane recruitment is essential for the phosphorylation of phosphoinositides, the cellular localization of PI3Kγ as a protein kinase remains unclear. It has merely been described that PI3Kγ protein kinase activity leading to MAPK activation seems to be restricted to a cytosolic localization. Here, we demonstrate that a hybrid-PI3Kγ having protein kinase, but not lipid kinase activity shows a similar cellular distribution with a high membrane association and comparable liposome binding behavior to wild-type PI3Kγ. Binding of PI3Kγ to liposomes mimicking the natural plasma membrane slightly stimulates autophosphorylation of PI3Kγ. However, liposomes containing an unphysiologically high amount of PI inhibit autophosphorylation of PI3Kγ. Finally, PI3Kγ bound to membrane fragments does not show autophosphorylation which is possibly due to protein-protein-interactions at the plasma membrane. This indicates that not only MAPK activation, but PI3Kγ protein kinase activity in general is localized in the cytosol.

Phosphoinositide 3-kinase gamma has multiple phospholipid binding sites.

Phosphoinositide 3-kinase gamma is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion constructs of human phosphoinositide 3-kinase gamma, we show that each domain is capable of binding anionic phospholipids to varying degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any single protein domain results in a complete loss of kinase activity toward both lipids and proteins.

Adsorption of GST-PI3Kgamma at the air-buffer interface and at substrate and nonsubstrate phospholipid monolayers.

The recruitment of phosphoinositide 3-kinase gamma (PI3Kgamma) to the cell membrane is a crucial requirement for the initiation of inflammation cascades by second-messenger production. In addition to identifying other regulation pathways, it has been found that PI3Kgamma is able to bind phospholipids directly. In this study, the adsorption behavior of glutathione S-transferase (GST)-PI3Kgamma to nonsubstrate model phospholipids, as well as to commercially available substrate inositol phospholipids (phosphoinositides), was investigated by use of infrared reflection-absorption spectroscopy (IRRAS). The nonsubstrate phospholipid monolayers also yielded important information about structural requirements for protein adsorption. The enzyme did not interact with condensed zwitterionic or anionic monolayers; however, it could penetrate into uncompressed fluid monolayers. Compression to values above its equilibrium pressure led to a squeezing out and desorption of the protein. Protein affinity for the monolayer surface increased considerably when the lipid had an anionic headgroup and contained an arachidonoyl fatty acyl chain in sn-2 position. Similar results on a much higher level were observed with substrate phosphoinositides. No structural response of GST-PI3Kgamma to lipid interaction was detected by IRRAS. On the other hand, protein adsorption caused a condensing effect in phosphoinositide monolayers. In addition, the protein reduced the charge density at the interface probably by shifting the pK values of the phosphate groups attached to the inositol headgroups. Because of their strongly polar headgroups, an interaction of the inositides with the water molecules of the subphase can be expected. This interaction is disturbed by protein adsorption, causing the ionization state of the phosphates to change.

Long chain CoA esters as competitive antagonists of phosphatidylinositol 4,5-bisphosphate activation in Kir channels.

Long chain fatty acid esters of coenzyme A (LC-CoA) are potent activators of ATP-sensitive (K(ATP)) channels, and elevated levels have been implicated in the pathophysiology of type 2 diabetes. This stimulatory effect is thought to involve a mechanism similar to phosphatidylinositol 4,5-bisphosphate (PIP2), which activates all known inwardly rectifying potassium (Kir) channels. However, the effect of LC-CoA on other Kir channels has not been well characterized. In this study, we show that in contrast to their stimulatory effect on K(ATP) channels, LC-CoA (e.g. oleoyl-CoA) potently and reversibly inhibits all other Kir channels tested (Kir1.1, Kir2.1, Kir3.4, Kir7.1). We also demonstrate that the inhibitory potency of the LC-CoA increases with the chain length of the fatty acid chain, while both its activatory and inhibitory effects critically depend on the presence of the 3'-ribose phosphate on the CoA group. Biochemical studies also demonstrate that PIP2 and LC-CoA bind with similar affinity to the C-terminal domains of Kir2.1 and Kir6.2 and that PIP2 binding can be competitively antagonized by LC-CoA, suggesting that the mechanism of LC-CoA inhibition involves displacement of PIP2. Furthermore, we demonstrate that in contrast to its stimulatory effect on K(ATP) channels, phosphatidylinositol 3,4-bisphosphate has an inhibitory effect on Kir1.1 and Kir2.1. These results demonstrate a bi-directional modulation of Kir channel activity by LC-CoA and phosphoinositides and suggest that changes in fatty acid metabolism (e.g. LC-CoA production) could have profound and widespread effects on cellular electrical activity.

Functions of sorting nexin 17 domains and recognition motif for P-selectin trafficking.

SNX17 is a member of the sorting nexin family (SNX), a group of hydrophilic proteins whose common characteristic property is a phox homology (PX) domain. The PX domain directs SNXs to phosphatidylinositides containing membranes of the endosomal compartment, where the SNXs are involved in the sorting of transmembrane proteins. SNX17 is known to interact with P-selectin and the LDL receptor family. Here, we report that the PX domain of SNX17 specifically binds to phosphatidylinositol 3-phosphate-containing membranes. The functional part of SNX17 that binds P-selectin or Patched (PTCH) consists of a truncated FERM domain and a unique C terminus together (FC-unit). In a yeast two-hybrid analysis a putative recognition motif for the FC-unit was revealed within P-selectin as FxNaa(F/Y). When HepG2 cells overexpress P-selectin together with SNX17, SNX17 changes its distribution from early endosomes to lysobisphosphatidic acid-containing late endosomes. Furthermore, overexpressed SNX17 restrains P-selectin in the outer membrane of the late endosomal compartment, thus preventing the normal lysosomal accumulation of P-selectin. These results suggest that the PX domain is necessary for the intracellular localisation, while the FC-unit is required for cargo recognition. We hypothesise that the expression level of SNX17 may regulate the lysosomal degradation, at least for P-selectin, by suppressing its entry into the inner vesicles of the multi-vesicular bodies (MVBs).

Signal transduction of c-Kit receptor tyrosine kinase in CHRF myeloid leukemia cells.

PURPOSE: The tyrosine kinase receptor c-Kit (stem cell factor receptor, CD117) is a potential target for signal transduction therapy in different cancers. In this study we investigated c-Kit in CHRF cells, a megakaryoblastic cell line of Acute Myeloid Leukemia (FAB M7). MATERIALS AND METHODS: We characterized the interactions between c-Kit and PI 3-kinase (p85) after stimulation with SCF (stem cell factor) as well as the regulation of SHP-1 and SHP-2 associated with Kit in this cell line. RESULTS: Stimulation with SCF leads to a significant increase in interaction between Kit and p85 as well as in receptor associated PI 3-kinase activity. Interestingly, using different kinds of substances (AG 1295, CGP 53716) to inhibit the tyrosine kinase activity of c-Kit blocked activation of c-Kit, but the association of p85 still increased after SCF stimulation even when the tyrosine kinase activity of the receptor was completely blocked. In contrast, the other known interaction partners of c-Kit, SHP-1 and SHP-2, exhibited a basal association with c-Kit and no change of the association could be detected after stimulation of CHRF cells with SCF or treatment with the kinase inhibitors. CONCLUSIONS: Therefore, we suggest that association of p85, SHP-1, and SHP-2 to c-Kit in CHRF cells can, at least in part, occur in a c-Kit kinase-activity independent manner. In contrast, the kinase activity of c-Kit is necessary for the activation of receptor-associated PI 3-kinase.