Development of a novel AAK1 inhibitor via Kinobeads-based screening.

A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and ß/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.

Transcriptional, biochemical, and immunohistochemical analyses of CaMKKß/2 splice variants that co-localize with CaMKIV in spermatids.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca2+-dependent physiological and pathophysiological pathways. Further, CaMKKß/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKß/2 splice variants (CaMKKß-3 and ß-3x). RT-PCR analyses revealed that mouse CaMKKß-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKß-3 and ß-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKß-3 or ß-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKß-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKß-1. We also observed the co-localization of CaMKKß-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKß-3 exhibited Ca2+/CaM-induced kinase activity similar to CaMKKß-1. Conversely, we noted that CaMKKß-3x impaired Ca2+/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKß-1 or ß-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKß-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKß-1 and ß-3. Collectively, CaMKKß-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.

Rapid detection of calmodulin/target interaction via the proximity biotinylation method.

Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity.

The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell-dependent monocytic cells with B cell-activating ability.

Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin ß receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

Substrate recognition by Arg/Pro-rich insert domain in calcium/calmodulin-dependent protein kinase kinase for target protein kinases.

Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+ -signaling pathways. Mammalian cells expressing CaMKKα and CaMKKß lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIα and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKKα and CaMKKß inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKIα at Thr177, HA-CaMKIV at Thr196, and HA-AMPKα at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKKα and CaMKKß interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors.

Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), a Ca2+/CaM-dependent enzyme that phosphorylates and activates multifunctional kinases, including CaMKI, CaMKIV, protein kinase B/Akt, and 5'AMP-activated protein kinase, is involved in various Ca2+-signaling pathways in cells. Recently, we developed an ATP-competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, Ohtsuka et al. Biochemistry 2020, 59, 1701-1710). To gain mechanistic insights into the interaction of CaMKK with TIM-063, we prepared TIM-063-coupled sepharose (TIM-127-sepharose) for association/dissociation analysis of the enzyme/inhibitor complex. CaMKKα/ß in transfected COS-7 cells and in mouse brain extracts specifically bound to TIM-127-sepharose and dissociated following the addition of TIM-063 in a manner similar to that of recombinant GST-CaMKKα/ß, which could bind to TIM-127-sepharose in a Ca2+/CaM-dependent fashion and dissociate from the sepharose following the addition of TIM-063 in a dose-dependent manner. In contrast to GST-CaMKKα, GST-CaMKKß was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST-CaMKKß without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKKα prevented the inhibitor from interacting with the catalytic domain as the GST-CaMKKα mutant (residues 126-434) lacking the regulatory domain (residues 438-463) interacted with TIM-127-sepharose regardless of the presence or absence of Ca2+/CaM. Furthermore, CaMKKα bound to TIM-127-sepharose in the presence of Ca2+/CaM completely dissociated from TIM-127-sepharose following the addition of excess EGTA. These results indicated that TIM-063 interacted with and inhibited CaMKK in its active state but not in its autoinhibited state and that this interaction is likely reversible, depending on the concentration of intracellular Ca2+.

Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase.

Ca2+/calmodulin-dependent protein kinase kinases (CaMKKα and ß) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His-CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG-CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST-CaMKKα/His-CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Ni-sepharose and was observed in a Ca2+/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG-CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling.

Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target.

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins.

To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKß) with a similar potency (Ki = 0.35 µM for CaMKKα, and Ki = 0.2 µM for CaMKKß) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 µM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase ß at Thr144 in HeLa cells.

Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5'AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKß is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKß is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKß in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKß has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKß is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKß by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKß-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKß is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

Interaction of S100A6 with Target Proteins In Vitro and in Living Cells.

S100A6 is a member of the EF-hand Ca2+-binding protein family, which plays important roles in a wide variety of Ca2+ signaling in the cells, as well as in pathophysiological conditions. Herein, we describe analytical protocols for evaluating the interaction of S100A6 with multiple target proteins in vitro, including biotinylated S100A6 overlay, glutathione-S-transferase (GST)-precipitation, surface plasmon resonance, and a GST-precipitation assay in living cells. These methods will elucidate the detailed molecular mechanisms of S100A6/target interactions and further improve our understanding of the physiological significance of S100A6-mediated Ca2+ signaling. Moreover, they may be used to evaluate other physical S100/target interactions.

Regulation of Ca2+/calmodulin-dependent protein kinase kinase ß by cAMP signaling.

BACKGROUND: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKß is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKß into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKß at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKß in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKß. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKß is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKß-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKß at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKß at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKß in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKß into Ca2+/CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKß-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKß signaling through regulatory phosphorylation.

Interleukin 34 (IL-34) cell-surface localization regulated by the molecular chaperone 78-kDa glucose-regulated protein facilitates the differentiation of monocytic cells.

Interleukin 34 (IL-34) constitutes a cytokine that shares a common receptor, colony-stimulating factor-1 receptor (CSF-1R), with CSF-1. We recently identified a novel type of monocytic cell termed follicular dendritic cell-induced monocytic cells (FDMCs), whose differentiation depended on CSF-1R signaling through the IL-34 produced from a follicular dendritic cell line, FL-Y. Here, we report the functional mechanisms of the IL-34-mediated CSF-1R signaling underlying FDMC differentiation. CRIPSR/Cas9-mediated knockout of the Il34 gene confirmed that the ability of FL-Y cells to induce FDMCs completely depends on the IL-34 expressed by FL-Y cells. Transwell culture experiments revealed that FDMC differentiation requires a signal from a membrane-anchored form of IL-34 on the FL-Y cell surface, but not from a secreted form, in a direct interaction between FDMC precursor cells and FL-Y cells. Furthermore, flow cytometric analysis using an anti-IL-34 antibody indicated that IL-34 was also expressed on the FL-Y cell surface. Thus, we explored proteins interacting with IL-34 in FL-Y cells. Mass spectrometry analysis and pulldown assay identified that IL-34 was associated with the molecular chaperone 78-kDa glucose-regulated protein (GRP78) in the plasma membrane fraction of FL-Y cells. Consistent with this finding, GRP78-heterozygous FL-Y cells expressed a lower level of IL-34 protein on their cell surface and exhibited a reduced competency to induce FDMC differentiation compared with the original FL-Y cells. These results indicated a novel GRP78-dependent localization and specific function of IL-34 in FL-Y cells related to monocytic cell differentiation.

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase ß.

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

Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target.

S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1-30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, A11, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1-30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function.

Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help.

To reconcile conflicting reports on the role of CD40 signaling in germinal center (GC) formation, we examined the earliest stages of murine GC B cell differentiation. Peri-follicular GC precursors first expressed intermediate levels of BCL6 while co-expressing the transcription factors RelB and IRF4, the latter known to repress Bcl6 transcription. Transition of GC precursors to the BCL6hi follicular state was associated with cell division, although the number of required cell divisions was immunogen dose dependent. Potentiating T cell help or CD40 signaling in these GC precursors actively repressed GC B cell maturation and diverted their fate towards plasmablast differentiation, whereas depletion of CD4+ T cells promoted this initial transition. Thus while CD40 signaling in B cells is necessary to generate the immediate precursors of GC B cells, transition to the BCL6hi follicular state is promoted by a regional and transient diminution of T cell help.

SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus.

Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSF1-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSF1-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, overexpression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSF1-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C-terminus-dependent manner.

Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen.

To search for novel target(s) of the Ca(2+)-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca(2+)/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca(2+)-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles.

Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKß phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼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/CaMKKß chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKß/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.