December 17, 2007

Unscrambling B-cell signals
RIKEN researchers uncover new roles for old players in a complex web of cross-talking
proteins important in immunity and cancer.
Coordination of cell functions is a bewilderingly complex process. In the latest issue of
The Journal of Experimental Medicine, scientists led by Tomohiro Kurosaki at RIKEN's
Research Center for Allergy and Immunology report findings that improve ourunderstanding of a key communication network within immune cells.
B cells are the key immune system agents that create antibodies. One way that B cells
can respond to external stimuli is through a surface protein called the B cell receptor(BCR). When a specific antigen (a molecule that stimulates an immune response) docks
in the BCR, sparking a signal transduction event, in which a cascade of biochemical
reactions among enzymes occurs that regulates immune responses, inflammation andcell survival.
One such signal transduction pathway involves the Nuclear factor-κB (NFκB) andinhibitor of Nuclear factor-κBkinase (IKK) families of proteins, which are major players incell signaling. As DNA-binding proteins regulating gene expression, the ubiquitous
NFκB proteins activate a large number of genes that eventually lead to complex
physiological and biochemical events, dramatically amplifying the original signal. WhenNFκB-signaling goes awry, various immune disorders and cancers may ensue. Hence,
the intricate molecular mechanisms by which BCR engagement is coupled to NFκB
activation have been subjected to intense scrutiny.
In all non-stimulated cells, NFκBs are kept in check by being bound to IKK proteins andsequestered outside the nucleus, away from the genes. When activated by various
stimuli, the IKKs are modified by phosphorylation - the addition of phosphate groups, byenzymes called kinases, to the side arms of amino acids, the building blocks of proteins,which leads to their degradation by the cell's house-keeping processes. The liberated
NFκBs can then enter the nucleus to regulate many genes.
Clearly, IKK - a complex of at least three protein kinases, IKKα, IKKβ and IKKγ represents a major control point, one affected by various adaptor proteins. Theseinclude CARMA1, a scaffolding protein that brings together Bcl10, a bridge-like protein,
and an effector protein, MALT1, into an assemblage (CBM complex) working in concert
to stimulate the IKK complex. For B-cells, PKCβ, another kinase, acts upstream of theCBM complex to activate IKK, but the exact molecular mechanisms involved, including
how CARMA1 becomes phosphorylated, and thus activated, to mobilize the assemblyof the CBM and IKK complexes, remain undefined. The RIKEN researchers focused onclarifying how this crucial piece fits into the intricate NFκB/IKK puzzle.
Kurosaki and colleagues used special chicken cells expressing BCR to explore thespecific regions of the CARMA1 protein important for IKK activation. These experiments
hinged on engineered antibodies that identify specifically phosphorylated amino acids,
allowing the scientists to eventually pinpoint critical CARMA1 amino acids that arephosphorylated during BCR engagement. By systematically deleting from these chicken
cells the activities of several suspected kinases, the researchers concluded that PKCβphosphorylated one of these CARMA1 amino acids, Ser668, whereas IKKβ wasresponsible for phosphorylating another amino acid, Ser578.
To confirm these results, the RIKEN group tested whether the CARMA1-directed
enzymatic deficiencies in these chicken cells could be rescued by adding external,active, sources of these specific enzymes into the cells. As expected, phosphorylationof Ser668 was restored by active PKCβ, while re-introduced, active, IKKβ
phosphorylated Ser578. IKKβ's role upstream of the IKK complex is particularlyintriguing. A biochemical examination verified that CBM complex assembly is dependent
on Ser578 phosphorylation. These findings were further supported with the significant
inhibition of CBM assembly observed in cells containing a CARMA1 mutant harboring adifferent amino acid in the Ser578 position.
The RIKEN scientists theorize that IKKβ plays more complex feedback roles inNFκB-signaling than previously believed. A deeper understanding of these criticalswitches will shape the development of therapeutic agents for autoimmune disorders
and cancers.
Original work:
Shinohara, H., Maeda, S., Watarai, H., Kurosaki, T. IκB kinase β-induced
phosphorylation of CARMA1 contributes to CARMA1-Bcl10-MALT1 complex formationin B cells. The Journal of Experimental Medicine, published online on December 17,

2007

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