Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5'-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N(6)-(1-methylbutyl)-ATP and genetically engineered CaMKKα mutant, CaMKKα (Phe(230)Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKKα (Phe(230)Gly), but not with wild-type kinase, in the presence of N(6)-(1-methylbutyl)-ATP and Ca(2+)/CaM, induced significant threonine phosphorylation of a 50kDa protein as well as CaMKI phosphorylation at Thr(177). The 50kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKKα and β isoforms at Thr(355)in vitro. Phosphorylation of HA-Syndapin I at Thr(355) in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca(2+)-signaling pathways that are independent of downstream protein kinases.

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) 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. CaMKKβ 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 CaMKKβ 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 CaMKKβ 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 CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-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 CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

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.

ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, which mediates stimulatory effects on ABCA1 gene expression, could interfere with the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) cascade. ABCA1 promoter activity was examined by reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation-loop phosphorylation (Thr(196)) of CaMKIV. We investigated the influence of the constitutively active form (CaMKIVc) or CaMKIV knockdown on ABCA1 expression. Increased abundance of ABCA1 protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Exendin-4 also stimulated ABCA1 promoter activity, but failed to do so in the presence of STO-609, a CaMKK inhibitor. Up-regulation of CaMKIV phosphorylation (at Thr(196)) peaked after 10 min. of exposure to exendin-4. CaMKIVc enhanced or up-regulated ABCA1 promoter activity in INS-1 cells. Furthermore, exendin-4 induction of ABCA1 protein expression was significantly suppressed in cells treated with CaMKIV-siRNA. Activation of the CaMKK/CaMKIV cascade by exendin-4 stimulated ABCA1 gene transcription, indicating that exendin-4 plays an important role in insulin secretion and cholesterol ester content in pancreatic beta cells.

We show that Ca(2+)/calmodulin(CaM)-dependent protein kinase I (CaMKI) is directly inhibited by its S-glutathionylation at the Cys(179). In vitro studies demonstrated that treatment of CaMKI with diamide and glutathione results in inactivation of the enzyme, with a concomitant S-glutathionylation of CaMKI at Cys(179) detected by mass spectrometry. Mutagenesis studies confirmed that S-glutathionylation of Cys(179) is both necessary and sufficient for the inhibition of CaMKI by diamide and glutathione. In transfected cells expressing CaMKI, treatment with diamide caused a reversible decrease in CaMKI activity. Cells expressing mutant CaMKI (179CV) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKI via its modification at Cys(179) is an important mechanism in processing calcium signal transduction in cells.

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 CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear.

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.

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.