A Novel Interaction between the Juxtamembrane Region of the p55 Tumor Necrosis Factor Receptor and Phosphatidylinositol-4-phosphate 5-Kinase

Abstract

Tumor necrosis factor-␣ (TNF-␣) binding to its receptors leads to a diversity of biological responses. The actions of TNF are the result of the interaction of cytoplasmic proteins that bind directly to the intracellular domains of the two TNF receptors, p55 and p75. Here we report a novel interaction between the juxtamembrane region of the p55 TNF receptor and a newly discovered 47-kDa isoform of phosphatidylinositol-4-phosphate 5-kinase (PIP5K), a member of the enzyme family that generates the key signaling messenger, phosphatidylinositol 4,5-bisphosphate. The interaction was found to be specific for the p55 TNF receptor and was not observed with the p75 TNF receptor, the Fas antigen, or the p75 neurotrophin receptor, which are other members of the TNF receptor superfamily. In vitro experiments using recombinant fusion proteins verify the authenticity of the interaction between the p55 receptor and PIP5KII␤, a new isoform of PIP5K, but not the previously identified 53-kDa PIP5KII␣. Treatment of HeLa cells with TNF-␣ resulted in an increased PIP5K activity. These results indicate that phosphatidylinositol turnover may be linked to stimulation of the p55 TNF receptor and suggest that a subset of TNF responses may result from the direct association of PIP5KII␤ with the p55 TNF receptor. Tumor necrosis factor-␣ (TNF-␣)1 initiates its proliferative, differentiative, or cytotoxic actions on mammalian cells by binding to two transmembrane molecules, the p55 and p75 TNF receptors (1-3). The p55 receptor is responsible for many of the biological effects of TNF, including programmed cell death, cell differentiation, and cell proliferation (4 -6). A major step in understanding the mechanism of the p55 receptor has been the identification of the interacting protein, TRADD, which accounts for signals leading to apoptosis and increased gene expression through NF-B-mediated events (7). Likewise, the p75 receptor is capable of signal transduction through the association of ring finger proteins, such as TRAF1 and TRAF2, with the cytoplasmic domain of p75 (8, 9).The interaction of TRADD with the p55 TNF receptor has revealed the importance of protein-protein interactions via a region of homology called the "death domain." This sequence has been found in a variety of transmembrane and cytosolic molecules and is usually localized at the C-terminal region of each protein. The functional significance of this domain has been demonstrated in studies with the p55 TNF receptor and the Fas antigen, which contain similar functional death domain sequences (10). The binding of p55 receptors to TRADD, a cytoplasmic protein containing a death domain, and the binding of the Fas antigen to FADD, an analogous protein, have been localized to an 80-amino acid region at the C terminus of both receptors. While overexpression of TRADD or FADD in heterologous cells leads to cell death (7, 11), deletions or mutations in the death domains abolish the ability of these molecules to participate in the initiation of apoptosis (6,12).A link to the interleuki...

Phosphorylation of 130- and 95-kDa Substrates Associated with Tumor Necrosis Factor-α Receptor CD120a (p55)

“…We specifically used an antagonistic monoclonal antiCD120a (p55) antibody to prevent artifactual cross-linking of CD120a (p55), pp130, pp95, and the associated Ser/Thr kinase activity during immunoprecipitation. These findings suggest that both proteins constitutively interact with CD120a (p55) in the absence of ligand, a conclusion supported in the case of pp130, by the finding of a constitutive association of 35 S-labeled pp130 with CD120a (p55). Although these findings contrast with observations made for some CD120a (p55)-associated proteins, including TRADD and RIP for which interaction with CD120a (p55) has been shown to occur in a ligand-dependent fashion (4,6), other proteins, especially those that bind to the membrane proximal region of the cytoplasmic domain, such as phosphatidylinositol 4,5-bisphosphate kinase, appear to interact in a constitutive fashion, similar to pp130 and pp95 (35).…”

Phosphoinositides in Constitutive Membrane Traffic

“…PIP 5-kinases were originally purified as activities that phosphorylated PtdIns(4)P to PtdIns (4,5)P 2 and subsequently cDNAs for six enzymes have been cloned (25,37,157,159,209). Based on sequence relationships, the PIP5Ks were grouped into two families called types I and II.…”

“…PIP4K␣ was originally purified as an activity that phosphorylated a commercial PtdIns(4)P preparation to PtdIns(4,5)P 2 (18,208) and was named PIP5K type II␣. After this and a related enzyme, PIP5K type II␤, were cloned (25,37,79), it was discovered that the enzymes actually phosphorylated the D-4 position (283). The actual substrate in the bovine brain PtdIns(4)P that was used for the original purification of the enzymes was a previously unidentified lipid, PtdIns(5)P. Thus these enzymes should be called PIP4Ks.…”

Phosphatidylinositol Phosphate Kinases, a Multifaceted Family of Signaling Enzymes

“…These include the p55 tumor necrosis factor (TNF) receptor and the epidermal growth factor (EGF) receptor (27,43). In the case of the p55 TNF receptor, only PIPKII␤ associates with the receptor; the highly homologous PIPKII␣ does not (27). The EGF receptor is reported to associate with both PI 4-kinase and PIP 5-kinase activities, although the kinase isoforms have not been defined (43).…”