A Kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury

© 2020, The Author(s). Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death. Our screen and validation studies reveal an essential role of Cdkl5-kinase in RTEC cell death. In mouse models, genetic or pharmacological Cdkl5 inhibition mitigates nephrotoxic and ischemia-associated AKI. We propose that Cdkl5 is a stress-responsive kinase that promotes renal injury in part through phosphorylation-dependent suppression of pro-survival transcription regulator Sox9. These findings reveal a surprising non-neuronal function of Cdkl5, identify a pathogenic Cdkl5-Sox9 axis in epithelial cell-death, and support CDKL5 antagonism as a therapeutic approach for AKI

Protein phosphatases that regulate multifunctional Ca2+/calmodulin-dependent protein kinases: from biochemistry to pharmacology

Pergamon Press, Ishida, Atsuhiko ; Shigeri, Yasushi ; Taniguchi, Takanobu ; Kameshita, Isamu, Pharmacology and Therapeutics, 100(3), 2003, 291-305. authorMultifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in Ca(2+) signaling pathways, such as the regulation of the neuronal functions of learning, memory, and neuronal cell death. The activities of the kinases are strictly regulated by protein phosphorylation/dephosphorylation. Although the activation mechanisms for multifunctional CaMKs through phosphorylation, which correspond to "switch on," have been extensively studied, the negative regulatory mechanisms through dephosphorylation, which correspond to "switch off," have not. In this review, we focused on the regulation of multifunctional CaMKs by the protein phosphatases responsible. We first summarized the current understanding of negative regulation of CaMKs by known protein phosphatases and their physiological significance. We then discussed newly developed methods for detection of protein phosphatases involved in the regulation of CaMKs. We also summarized the biochemical properties of a novel protein phosphatase, which we isolated with the new methods and designated as CaMK phosphatase (CaMKP), and its homologue. Pharmacological implications for neuronal functions including memory and neuronal cell death are discussed from the viewpoint that regulation of protein kinase activity can be elucidated by focusing on protein phosphatases involved in its "switch off" mechanism

Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV

Elsevier, Ishida, A. ; Kameshita, I. ; Okuno, S. ; Kitani, T. ; Fujisawa, H., ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 407(1), 2002, 72-82 authorCalmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly--Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase α, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 μM CaM was about 10 μg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly--Arg could partially substitute for poly(Lys), but protamine, spermine, and poly--Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The ^P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr^. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca^/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM

Stimulation of Ca^<2+>/calmodulin-dependent protein kinase phosphatase by polycations

Elsevier, Ishida, A. ; Kameshita, I. ; Kitani, T. ; Okuno, S. ; Takeuchi, M. ; Fujisawa, H., ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 408(2), 2002, 229-238Ca^/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca^/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly--lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101–109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn^. Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity

Generation of a polyclonal antibody that simultaneously detects multiple Ser/Thr protein kinases

Elsevier, Isamu Kameshita, Shun Kinoshita, Yasushi Shigeri, Yoshiro Tatsu, Noboru Yumoto, Atsuhiko Ishida, JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, 60(1), 2004, 13-22 authorIn order to obtain a polyclonal antibody that recognizes various protein kinases, a peptide corresponding to an amino acid sequence of a highly conserved subdomain (subdomain VIB) of the protein kinase family was synthesized and used for immunization. When the synthetic peptide, CVVHRDLKPENLLLAS, was coupled to keyhole limpet hemocyanin (KLH) and used to immunize rabbits, polyclonal antibodies that detected multiple protein kinases on a Western blot were generated. One of the antibodies obtained, KI98, detected a variety of purified Ser/Thr protein kinases, such as calmodulin-dependent protein kinase II (CaM-kinase II), calmodulin-dependent protein kinase IV (CaM-kinase IV), cAMP-dependent protein kinase, protein kinase C, and Erk2. The antibody detected as low as 0.2 ng of protein kinases blotted onto a nitrocellulose membrane by dot-immunobinding assay. When a rat brain extract was analyzed with this antibody, various protein kinases were simultaneously detected. The present anti-peptide antibody with a broad spectrum of cross-reactivity to multiple protein kinases may be a powerful tool for comprehensive analysis focused on protein kinases

Identification of major Ca^<2+>/calmodulin-dependent protein kinase phosphatase-binding proteins in brain. Biochemical analysis of the interaction.

Elsevier, Ishida, A. ; Tada, Y. ; Nimura, T. ; Sueyoshi, N. ; Katoh, T. ; Takeuchi, M. ; Fujisawa, H. ; Taniguchi, T. ; Kameshita, I., ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 435(1), 2005, 134-146. authorCa^/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca^/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP–glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP–GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH

Inhibitors of the Ca^<2+>/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

authorCa^/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca^/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro