In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5′-triphosphate as a phosphate donor
© Yurimoto et al.; licensee BioMed Central Ltd. 2012
Received: 25 September 2012
Accepted: 29 November 2012
Published: 5 December 2012
Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases — including CaMKI, CaMKIV, and AMPK— to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKIα (Thr177) and of AMPK (Thr172) in vitro. Kinetic analysis indicated that the Km values of CaMKK isoforms for GTP (400-500 μM) were significantly higher than those for ATP (~15 μM), and a 2- to 4-fold decrease in Vmax was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of ~45 kDa and ~35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.
KeywordsCalmodulin CaMKK Phosphate donor GTP Phosphorylation
Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) has been classified as a novel member of the calmodulin kinase (CaMK) family that specifically phosphorylates a single Thr residue (Thr177 or Thr196, respectively) within the activation loop in each of two multifunctional calmodulin (CaM) kinases, CaMKI and CaMKIV; these phosphorylation events cause a large increase in catalytic efficiency [1–3]. Accumulated biochemical evidence indicates that CaMKK phosphorylates Akt/Protein kinase B  and AMPK (AMP-activated protein kinase) family members including the catalytic subunit of AMPK (AMPKα) at Thr172 [5–8] and SAD-B (known as a brain-specific kinase, BRSK1) at Thr189 ; either phosphorylation event causes significant catalytic activation, and these findings indicate that CaMKK confers Ca2+ dependence on other signalling pathways. In mammals, two CaMKK genes (CaMKKα and CaMKKβ) have been identified, and both are highly expressed in the brain; the α isoform is also expressed in various peripheral tissues such as thymus and spleen . A CaMKK gene has been found in Caenorhabditis elegans and in Aspergillus nidulans, and the proteins encoded by these genes are components of the CaMK cascade within the respective organisms [11, 12]. Interestingly, both mammalian CaMKK isoforms bind to Ca2+/CaM as well as to CaMKI and CaMKIV proteins that function downstream of Ca2+/CaM complexes [13, 14]. Indeed, Ca2+/CaM binding is absolutely required for the relief of CaMKKα autoinhibition , which results in its activation like other CaMKs. Previous structural and functional studies of CaMKK have revealed a novel CaM-binding motif (1-16) , and the unique feature of the CaM-binding segment in CaMKK is required for the autoinhibitiory mechanism through Ile441 in CaMKKα .
Many cell types depend on a functional CaM-kinase cascade that leads to activation of CaMKI and CaMKIV in response to Ca2+ mobilization. The CaMKK/CaMKIV cascade has an important role in Ca2+-dependent regulation of gene expression that is mediated by phosphorylation of transcription factors such as cAMP-response element binding protein (CREB) . Analysis of CaMKIV-deficient mice revealed that the CaMKIV-mediated pathway plays an important role in the development and function of the cerebellum and is critical for both male and female fertility [18, 19]. The CaMKK/CaMKI cascade has been shown to be involved in various neuronal functions, including spinogenesis , dendritic arborization  and cortical axon elongation . Recent accumulated data have shown that Ca2+-dependent phosphorylation and consequent activation of AMPK is mediated by CaMKKβ when T-cells are activated via the antigen receptor  or when HeLa cells were treated with a Ca2+ ionophore . Based on these results, CaMKK is predicted to act as a regulatory protein kinase in various Ca2+-dependent cellular processes in vivo. Therefore, identification of novel target(s) of CaMKK is important for the clarification of the CaMKK-mediated signaling pathway. Here, we found and characterized an enzymatic feature of CaMKK isoforms that might be useful in screens for novel targets of CaMKK; specifically, these enzymes can use GTP, as well as ATP, as a phosphate donor.
Results and discussion
CaMKK is capable of using Mg-GTP as a phosphate donor
Isoform specificity of CaMKK for utilizing Mg-GTP
Kinetic analysis of CaMKK isoforms
Kinetic Parameters of CaMKK Isoforms
V max / K m
Inhibition of CaMKK activities using Mg-GTP by STO-609
Detection of phosphorylation of CaMKK targets in rat brain extracts
Searching for target substrates is always important to evaluate the physiological significance of a protein kinase. Since 1995, when CaMKK was cloned as an activator for CaMKI and CaMKIV , we have attempted to develop methods using enzyme-substrate interactions and an ATP-analogue to search for novel CaMKK targets; in that time, we found two potential CaMKK targets — SAD-B  and Syndapin 1 . Here, we attempted to examine various nucleotides as phosphate donors for in vitro phosphorylation of target substrates by CaMKKs. We have shown that CaMKK isoforms were capable of using Mg-GTP as a phosphate donor. Usage of this nucleotide for CaMKK activity varies depending on downstream targets. 1) Both CaMKK isoforms were capable of phosphorylating CaMKIα (at Thr177) with Mg-GTP, as well as with Mg-ATP. 2) AMPKα can be phosphorylated (at Thr172) with Mg-GTP only by CaMKKβ, not by CaMKKα. Although very few protein kinases are known to use GTP as well as ATP, CaMKK is not the first protein kinase to use both nucleotides. Casein kinase II has been well characterized in its ability to use GTP and ATP [26, 28, 32]. Very recently, Drosophila and rat CaMKIIα have been shown to utilize GTP for exogenous substrate phosphorylation and autophosphorylation in vitro . In addition, previous reports have shown that four mammalian serine/threonine protein kinases — including protein kinase Cδ, Nercc1, mst-3 and AGT (O6-alkylguanine-DNA alkyltransferase) kinase — are able to use GTP as a substrate [27, 33–35]. However, the physiological significance of GTP-dependent phosphorylation has been unexplored because ATP has been recognized as the only biologically relevant phosphate donor for protein kinases. In vitro, we found that the phosphorylation of a CaMKK target protein, specifically CaMKI, was induced by incubation of rat brain extract with recombinant CaMKKs in the presence of Mg-GTP and Ca2+/CaM, and that this phosphorylation was inhibited by STO-609, a CaMKK inhibitor. Based on these results, we suggest that this enzymatic feature of CaMKKs, specifically the ability to use GTP or UTP in place of ATP as phosphate donors in vitro, might be useful in screens for novel CaMKK targets. However, careful consideration should be required to use Mg-GTP as a phosphate donor for CaMKK isoforms, since the differential effect of Mg-GTP on the activities of CaMKK isoforms (Figure 2B). Furthermore, the specificity of the CaMKK-mediated phosphorylation reaction could be confirmed by addition of the CaMKK inhibitor, STO-609, even though some endogenous protein kinases have been shown to be capable of using GTP as a phosphate donor in vitro. Indeed, we detected two potential CaMKK target proteins with molecular weight of ~45 kDa and ~35 kDa whose phosphorylation was induced by incubation of rat brain extract with Mg-GTP and Ca2+/CaM and was inhibited by STO-609. Based on the molecular weight of those phosphoproteins on SDS-PAGE, ~35 kDa phosphoprotein might be a member of CaMKI isoforms (Figure 4A). However, among various known CaMKK target kinases including CaMKI, CaMKIV, PKB, AMPK, and SAD-B [1–9], CaMKK target with a molecular weight of ~45 kDa on SDS-PAGE has not been identified. Further study to identify these putative CaMKK substrates is absolutely required to evaluate novel CaMKK-mediated signaling pathway. According to our study, in vitro phosphorylation assays using GTP in combination with STO-609 are expected to be a useful method for detecting CaMKK substrates and assessing its function(s) in various tissue and cells.
Recombinant CaMKKα and β were expressed in and purified from Escherichia coli as described previously . Recombinant rat CaM was expressed in E. coli strain BL-21 (DE3) using the pET-CaM plasmid (kindly provided by Dr. Nobuhiro Hayashi, Fujita Health University, Toyoake, Japan) and then purified by phenyl-Sepharose column chromatography . Mutant recombinant rat CaMKIα (1-293, K49E) was expressed in E. coli strain JM-109 as a GST-fusion protein and purified by glutathione Sepharose column chromatography . Recombinant AMPK was expressed in E. coli strain BL21-CodonPlus (DE3) (Stratagene, La Jolla, CA) using the tricistronic pγ1β1His-α1 plasmid (kindly provided by Dr. Dietbert Neumann, Swiss Federal Institute of Technology, Zurich, Switzerland) and purified by Ni-NTA agarose column chromatography (Qiagen, Hilden, Germany) . Rabbit IgG antibodies against AMPKα and those against phospho-AMPKα at Thr172 were purchased from Cell Signaling Technology, Inc. (Danvers, MA). An anti-CaMKI antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An anti-GST antibody and an anti-phospho-threonine antibody were purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK) and from Invitrogen (Carlsbad, CA), respectively. An anti-phospho-CaMKI (phospho-Thr177) monoclonal antibody was generated as described previously . STO-609 was synthesized as described previously . ATP, GTP, UTP and CTP were purchased from Roche Applied Science (Indianapolis, IN).
In vitro assay for CaMKK activity
Purified recombinant CaMKKs (CaMKKα, 0.9 μg/ml; CaMKKβ, 1.7 μg/ml) were incubated individually with GST-CaMKIα (1-293, K49E, 0.5 mg/ml) or AMPK (0.5 mg/ml) at 30°C for one of several defined time periods in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(CH3COO)2, 1 mM DTT, 1 mM CaCl2 (2 mM in Figure 3), and 10 μM CaM (4 μM in Figure 3) in the presence of either 1 mM nucleotide or one of several defined concentrations between 50 and 400 μM of either [γ-32P]ATP (1,200 – 10,000 cpm/pmol) or [γ-32P]GTP (800 – 7,000 cpm/pmol); one of several defined concentrations of STO-609 (0-10 μg/ml in dimethyl sulfoxide at a final concentration of 4%) was included in individual reactions. Each reaction was initiated by the addition of cold nucleotide or [γ-32P]ATP or [γ-32P]GTP and terminated by addition of an equal volume of 2 x SDS-PAGE sample buffer; each terminated reaction was then subjected to SDS-PAGE or to spotting of aliquots (20 μl) onto phosphocellulose paper (Whatman P-81). These spotted phosphocellulose papers were then washed several times with 75 mM phosphoric acid. Phosphate incorporation into GST-CaMKIα (1-293, K49E) was determined using western blots generated with the gels and an anti-phospho-CaMKI antibody or using the spotted filters that were subjected to liquid scintillation counts.
Phosphorylation within rat brain extract
Rat brain samples were homogenized with 5 volumes of homogenization buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, 1% NP-40); homogenates were then centrifuged at 30,190 x g at 4°C for 30 min. The supernatant was stored at -80°C until use. Rat brain extract was incubated in a solution (100 μl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(CH3COO)2, 2 mM DTT, 2 mM CaCl2, and CaM, 0.5 μM okadaic acid; each sample was incubated in the absence or presence of either 0.1 mM or 1 mM GTP at 30°C for 1 or 30 min with or without recombinant CaMKKs for indicated time periods. Each reaction was terminated by addition of 20 μl of SDS-PAGE sample buffer followed by western blot analysis with either anti-phospho-CaMKI antibody and anti-CaMKI antibody or anti-phospho-threonine antibody.
Staining of western blots was performed with horseradish peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG (GE Healthcare UK Ltd., Buckinghamshire, UK), or anti-goat IgG antibody (Sigma, Saint Louis, MO) as a secondary antibody and chemiluminescence reagent (PerkinElmer Life Sciences, Waltham, MA) for signal detection. The intensity of the immunoreactive band was measured by densitometric scanning of scientific imaging film (KODAK BioMax Light Film, Carestream Health, Inc., Rochester, NY) for detection and measurement of chemiluminescence. Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA) was used to estimate protein concentration; bovine serum albumin was used as the protein standard.
Ca2+/CaM-dependent protein kinase
Ca2+/CaM-dependent protein kinase kinase
5′AMP-activated protein kinase
We thank Momoko Nitta (Kagawa University) for excellent technical assistance. Saki Yurimoto is a Research Fellow of Japan Society for the Promotion of Science (22-1342).
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