28 research outputs found
The Transmembrane Region Is Responsible for Targeting of Adaptor Protein LAX into âHeavy Raftsâ
BACKGROUND: The importance of membrane compartmentalization into specific membrane microdomains has been shown in many biological processes such as immunoreceptor signaling, membrane trafficking, pathogen infection, and tumor progression. Microdomains like lipid rafts, caveolae and tetraspanin enriched microdomains are relatively resistant to solubilization by some detergents. Large detergent-resistant membrane fragments (DRMs) resulting from such membrane solubilization can be conveniently isolated by density gradient ultracentrifugation or gel filtration. Recently, we described a novel type of raft-like membrane microdomains producing, upon detergent Brij98 solubilization, "heavy DRMs" and containing a number of functionally relevant proteins. Transmembrane adaptor protein LAX is a typical "heavy raft" protein. The present study was designed to identify the molecular determinants targeting LAX-derived constructs to heavy rafts. METHODOLOGY/PRINCIPAL FINDINGS: We prepared several constructs encoding chimeric proteins containing various informative segments of the LAX sequence and evaluated their effects on targeting to heavy rafts. Replacement of the polybasic membrane-proximal part of LAX by CD3Δ-derived membrane-proximal part had no effect on LAX solubilization. Similarly, the membrane-proximal part of LAX, when introduced into non-raft protein CD25 did not change CD25 detergent solubility. These results indicated that membrane-proximal part of LAX is not important for LAX targeting to heavy rafts. On the other hand, the replacement of transmembrane part of CD25 by the transmembrane part of LAX resulted in targeting into heavy rafts. We also show that LAX is not S-acylated, thus palmitoylation is not involved in LAX targeting to heavy rafts. Also, covalent dimerization was excluded as a cause of targeting into heavy rafts. CONCLUSIONS/SIGNIFICANCE: We identified the transmembrane domain of LAX as a first motif targeting transmembrane protein constructs to detergent-resistant heavy rafts, a novel type of membrane microdomains containing a number of physiologically important proteins
Systematic analysis of the ILâ17 receptor signalosome reveals a robust regulatory feedback loop
Mutation landscape of multiple myeloma measurable residual disease:identification of targets for precision medicine
Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors
Summary RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor ÎșB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers
Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling
RIPK2 mediates inflammatory signaling by the bacteria-sensing
receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a
proposed strategy to ameliorate NOD-mediated pathologies. Here,
we reveal that RIPK2 kinase activity is dispensable for NOD2
inflammatory signaling and show that RIPK2 inhibitors function
instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination
of RIPK2. We map the XIAP binding site on RIPK2 to the loop
between b2 and b3 of the N-lobe of the kinase, which is in close
proximity to the ATP-binding pocket. Through characterization of a
new series of ATP pocket-binding RIPK2 inhibitors, we identify the
molecular features that determine their inhibition of both the
RIPK2-XIAP interaction, and of cellular and in vivo NOD2 signaling.
Our study exemplifies how targeting of the ATP-binding pocket in
RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction
for modulation of NOD signaling
Palmitoylation of LAX-LAX-LAX protein.
<p>Protein acylation of the LAX-LAX-LAX protein construct was examined using the acyl-biotinyl exchange chemistry-based method as described in Experimental procedures. Lysate that was not treated with NH<sub>2</sub>OH (hydroxylamine) served as a negative control. A palmitoylated heavy raft protein, transferrin receptor (TfR, CD71), was used as a positive control. IP: SA, material isolated on streptavidin beads.</p
Schematic representation of the chimeric proteins used in this study.
<p>EC â extracellular part, TM â transmembrane part, SM â membrane-proximal part, IC â intracellular part, OFP â orange fluorescent protein, N â N-terminus of the protein.</p
Lack of covalent dimerization of the chimeric protein constructs.
<p>Transfectant cell lines expressing the indicated constructs were analyzed by SDS-PAGE under reducing or non-reducing conditions, followed by immunoblotting. Jurkat cells were used as a control expressing endogenous LAT. Positions of m.w. standards (in kDa) are indicated.</p
The effect of the transmembrane segment of LAX on targeting to heavy rafts.
<p>Membranes isolated from cells expressing the indicated constructs were solubilized in Brij98, subjected to gel filtration on Sepharose 4B, and analyzed by immunoblotting as described in legend to Fig. 2.</p