Role of IκB kinase β in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex

Abstract

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKβ is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKβ regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKβ regulates membrane fusion and the remodeling of the CBM complex formation.

Keywords: CBM complex; IKKβ; SNARE machinery; Ubiquitination.

Figure 1. PF4-Cre/IKKβ^flox/flox mice have defects in platelet in membrane fusion

(A) IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were stimulated with 0.1 U/mL thrombin for 3 min. Platelet samples were fixed and processed for electron microscopy (EM) analysis. The samples were analyzed by transmission EM and images were obtained using Gatan software, with the scale bars indicated (n=3). (B) Individual platelet granules in the resting and stimulated (IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox) platelets were counted and presented as a bar diagram.

Figure 2. CBM complex forms in activated platelets

(A) Extracts from resting human and mouse platelets were subjected to immunoblotting with anti-CARMA1, anti-MALT1, anti-Bcl10, anti-TAK1, anti-TAB2 and TRAF6 antibodies (n=3). Human (B) and mouse (C) platelets were stimulated with thrombin (0.025 U/mL) for 3 min. Platelet lysates were precleared and then incubated with anti-MALT1. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antibodies to MALT1, Bcl10 and CARMA1 (n=3).

Figure 3. Bcl10 is phosphorylated, and IKKβ plays a critical role in CBM complex formation, in platelets

(A) Mouse platelets were stimulated with thrombin (0.1 U/mL), collagen (2 μg), TRAP4 (1 μM), A23187 (1 μM) and PMA (100 nM) in a time-dependent manner, before being subjected to immunoblotting with anti-pBcl10 antibody (n=3). (B) Phosphorylated Bcl10 bands were denstiometrically analyzed. (C) Washed IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were activated by 0.1 U/mL thrombin and lysed. Lysates were precleared and then incubated with anti-MALT1. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antibodies to CARMA1, Bcl10, pBcl10, IKKβ, and pIKKβ as indicated (n=3). (D) Washed IKKβ^flox/flox platelets were preincubated with the Bcl10 inhibitor (NBP2-29323, 10 μM for 5 min), before being activated by 0.1 U/mL thrombin and subjected to immunoblotting using antibodies IKKβ and pIKKβ (n=3). (E) Washed IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were preincubated with the PKCδ inhibitor (rottlerin, 10 μM for 5 min), before being activated by 0.1 U/mL thrombin and lysed. Platelet lysates were precleared and then incubated with anti-Bcl10. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antibodies to Bcl10, pBcl10, CARMA1 and IKKα as indicated (n=3). (F) Washed IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were preincubated with a general PKC inhibitor (RO-31-8220, 1 μM for 5 min), and a PKCα/β inhibitor (Gö6976, 1 μM, for 5 min), before being activated by 0.1 U/mL thrombin and subjected to immunoblotting using antibodies against Bcl10 and pBcl10 (n=3). (G) Washed IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were preincubated with a PKCδ inhibitor (Rottlerin, 10 μM for 5 min) and, before being activated with 100 nM PMA, and subjected to immunoblotting using antibodies against Bcl10 and pBcl10 (n=3).

Figure 4. CBM complex formation, Bcl10 phosphorylation and Bcl10-induced IKKγ ubiquitination are defective in the absence of IKKβ, in stimulated platelets

Washed IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were activated by 0.1 U/mL thrombin and lysed. Platelet lysates were precleared and then incubated with anti-MALT1 (A) or anti-Bcl10 (B). Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antibodies to MALT1, CARMA1, IKKβ, pIKKβ and pERK as indicated (A; n=3) or antibodies to MALT1, CARMA1, Bcl10 and pBcl10 as indicated (B; n=3). (C) Washed IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were activated by 0.1 U/mL thrombin and lysed. Platelet lysates were precleared and then incubated with anti-IKKγ. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antibodies to ubiquitin, IKKγ, Bcl10, and pBcl10 as indicated (n=3). (D) Washed IKKβ^flox/flox or PF4-Cre/IKKβ^flox/flox platelets were activated by 0.1 U/mL thrombin and lysed. Platelet lysates were precleared and then incubated with anti-Bcl10. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antibodies to ubiquitin, and Bcl10 as indicated (n=3).