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MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

Karim ZA, Vemana HP, Khasawneh FT - PLoS ONE (2015)

Bottom Line: In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex.It was also found to regulate thrombogenesis and physiologic hemostasis.Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA, 91766, United States of America.

ABSTRACT
We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

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MALT1 is ubiquitinated in thrombin stimulated platelets.Platelets were stimulated with thrombin (0.025 U/mL) for different time points (sec) as indicated in the figure. Platelet lysates were precleared and then incubated with anti-MALT1. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using anti-MALT1 and anti-ubiquitin antibodies.
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pone.0119363.g001: MALT1 is ubiquitinated in thrombin stimulated platelets.Platelets were stimulated with thrombin (0.025 U/mL) for different time points (sec) as indicated in the figure. Platelet lysates were precleared and then incubated with anti-MALT1. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using anti-MALT1 and anti-ubiquitin antibodies.

Mentions: MALT1 acts not only as a scaffold protein but also as a protease that activates IKK complex in nucleated cells[29], and was shown, in nucleated cell systems such as mast cells, to be heavily polyubiquitinated. To determine whether platelets contain a functional proteasome system, we investigated whether MALT1 is modified by ubiquitin in platelets, under agonist stimulation conditions. Thus, platelets were stimulated with thrombin and MALT1 was immunoprecipitated, under denaturing conditions, and analyzed for its polyubiquitination state, in a time-dependent manner. Indeed, the “modified” MALT1 in the high-molecular-weight fraction was detected by an anti-ubiquitin antibody (Fig. 1), thereby providing evidence that platelets do have a functional proteasome system.


MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

Karim ZA, Vemana HP, Khasawneh FT - PLoS ONE (2015)

MALT1 is ubiquitinated in thrombin stimulated platelets.Platelets were stimulated with thrombin (0.025 U/mL) for different time points (sec) as indicated in the figure. Platelet lysates were precleared and then incubated with anti-MALT1. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using anti-MALT1 and anti-ubiquitin antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352082&req=5

pone.0119363.g001: MALT1 is ubiquitinated in thrombin stimulated platelets.Platelets were stimulated with thrombin (0.025 U/mL) for different time points (sec) as indicated in the figure. Platelet lysates were precleared and then incubated with anti-MALT1. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using anti-MALT1 and anti-ubiquitin antibodies.
Mentions: MALT1 acts not only as a scaffold protein but also as a protease that activates IKK complex in nucleated cells[29], and was shown, in nucleated cell systems such as mast cells, to be heavily polyubiquitinated. To determine whether platelets contain a functional proteasome system, we investigated whether MALT1 is modified by ubiquitin in platelets, under agonist stimulation conditions. Thus, platelets were stimulated with thrombin and MALT1 was immunoprecipitated, under denaturing conditions, and analyzed for its polyubiquitination state, in a time-dependent manner. Indeed, the “modified” MALT1 in the high-molecular-weight fraction was detected by an anti-ubiquitin antibody (Fig. 1), thereby providing evidence that platelets do have a functional proteasome system.

Bottom Line: In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex.It was also found to regulate thrombogenesis and physiologic hemostasis.Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA, 91766, United States of America.

ABSTRACT
We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

Show MeSH
Related in: MedlinePlus