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

Abstract

Ca 2+ /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 Ca 2+ -dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca 2+ /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.

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

“…1D, the first and fifth lanes from left) due to the intramolecular autophosphorylation of CaMKKs [29]. Mobility shifts of the enzymes were completely abolished in the presence of the CaMKK inhibitor, TIM-063 (26 µM) [30], indicating that DRP mutants of both CaMKK isoforms were catalytically active in a similar manner to each wild-type CaMKK, which is consistent with our previous observation in recombinant CaMKKa expressed in Escherichia coli [20].…”

“…4B). After incubation with 26 µ m a CaMKK inhibitor, TIM‐063 [30] for 6 h to suppress endogenous CaMKK activity, the cells were stimulated with 1 µ m ionomycin for 5 min, followed by immunoblot analyses with either an anti‐pCaMKIα at Thr177 (Fig. 4A, upper panel) or anti‐pCaMKIV at Thr196 (Fig.…”

“…Phosphorylation of each substrate was estimated by densitometric scanning of the immunoblots. Autophosphorylation of His–CaMKKα and β including ΔRP mutants was measured at 30 °C for 15 and 30 min in 20 µL of a solution containing 0.1–0.2 µg CaMKK isoforms, 50 m m HEPES pH 7.5, 10 m m Mg (CH 3 COO) 2 , 1 m m DTT, 2 m m CaCl 2 , 6 µ m CaM with or without 100 µ m ATP in the absence or presence of 26 µ m CaMKK inhibitor, TIM‐063 [30]. The reaction was terminated by the addition of SDS/PAGE sample buffer followed by immunoblot analysis using an anti‐CaMKKα or β antibody.…”

“…1C were incubated with corresponding substrates (GST-CaMKIa, GST-CaMKIV or AMPK: 10 µg) and 2 mM CaCl 2 /6 µM CaM in the absence (0 min,in insert panels) or presence of 100 µM ATP for the indicated time periods (2-30 min) followed by immunoblot analyses (insert panels) using anti-phosphoCaMKIa (at Thr177) (A), anti-phosphoCaMKIV (at Thr196) (B), and anti-phosphoAMPKa (at Thr172) (C) antibodies as described in "Materials and methods." The phosphorylation level of each substrate was quantitated by densitometric scanning of the immunoblot (insert) and expressed as a percentage of the average value of the 10-min time point (panel A) or the 30 AMPK kinase [18], inserted with the CaMKK RPdomain sequences. His 6 -tagged mutants of LKB1 were constructed in which the 86Arg-89Pro sequences in kinase subdomains II and III were replaced by the RP-domain sequences of the CaMKK isoforms (165Leu-190Leu in rat CaMKKa and 201Ile-226Arg in rat CaMKKb) (Fig.…”

Stabilization of Protein–Protein Interactions between CaMKK2 and 14–3–3 by Fusicoccins

“…CaMKK-mediated Ca 2+ signaling plays a key role in metabolic processes, inflammation, and cancer cell growth. Accordingly, CaMKKs are considered promising therapeutic targets for various diseases. − Yet, despite considerable efforts, no selective CaMKK inhibitor has been developed so far. Therefore, the stabilization of PPIs between CaMKK and 14–3–3 by small molecule compounds may be a plausible alternative and/or complementary strategy for inhibiting CaMKK activity.…”