The Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5′-AMP–activated protein kinase (AMPK) phosphorylation cascade affects various Ca²⁺-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca²⁺ concentrations. Moreover, the Ca²⁺/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca²⁺/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr¹⁴⁴ phosphorylation by activated AMPK converts CaMKKβ into a Ca²⁺/CaM-dependent enzyme as shown by completely Ca²⁺/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471–587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr¹⁴⁴ phosphorylation–dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr¹⁴⁴ antibody revealed phosphorylation of Thr¹⁴⁴ in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca²⁺-dependent AMPK activation by CaMKKβ.

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V_H17.2.25 (V_HT)-encoded H chain and the λ1 or λ2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had ∼2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V_HT/λ2 B cells significantly predominated over V_HT/λ1 B cells in MZ-(V_HT/λ1:V_HT/λ2 ≈ 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of λ2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V_HT/λ2 B cells were further augmented by doubling the V_HT gene, but dampened by the dysfunction of Bruton’s tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V_HT/λ1 and V_HT/λ2 IgM mAbs revealed a polyreactive nature of the V_HT/λ2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.

In germinal centers (GCs), B cells are selected through interaction with follicular dendritic cells bearing immune complexes and follicular helper T (Tfh) cells secreting Tfh cytokines, including IL-21. To analyze these cellular interactions, we have explored culture conditions that can simulate GC B cell selection in vitro using a mouse follicular dendritic cell line, FL-YB. FL-YB cells efficiently enhanced viability of cocultured mouse B cells in a BAFF-dependent fashion. Interestingly, we found that addition of IL-21, a major Tfh cytokine, readily induced death of B cells that were cocultured with FL-YB cells, whereas IL-21 alone sustained viability of B cells in the absence of FL-YB cells. The IL-21–induced death was dependent on a low m.w. soluble factor that was released from FL-YB cells, which was finally identified as PGE₂. Treatment of B cells with IL-21 plus PGE₂, but not either alone, resulted in enhanced expression of a proapoptotic protein Bim and the upstream transcription factor Foxo1. A PGE₂ receptor isoform, EP4, was responsible for IL-21/PGE₂–induced B cell death. Thus, PGE₂ is an endogenous chemical mediator that can switch pleiotropic actions of IL-21 on B cells. IL-21/PGE₂–induced B cell death was rescued if B cells were costimulated via CD40. In immunized mice, deficiency of IL-21R in B cells led to a significant decrease in the frequency of activated caspase-3–positive GC B cells concomitant with impaired affinity maturation of Abs. Taken together, results implicate a physiological role of IL-21/PGE₂–induced B cell death in GC B cell selection.

Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr¹⁷² in the AMPKα subunit more efficiently than CaMKKα, with a lower K_m (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu³⁵⁸ in CaMKKβ/Ile³²² in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.

Somatic hypermutation (SHM) of Ig variable region ( IgV ) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.