Yuki Shiro, Syouichi Katayama, Haruka Tsukamoto and Tetsuo Yamazaki : Pro-cathepsin D prevents aberrant protein aggregation dependent on endoplasmic reticulum protein CLN6., Molecular Genetics and Metabolism, Vol.143, No.1-2, 108539, 2024.
(要約)
We previously expressed a chimeric protein in which the small heat-shock protein αB-crystallin (αBC) is fused at its N-terminus to the C-terminus of the first transmembrane segment of the endoplasmic reticulum (ER) protein mitsugumin 23 and confirmed its localization to the ER. Moreover, overexpression of this N-terminally modified αBC was shown to prevent the aggregation of the coexpressed R120G αBC variant, which is highly aggregation-prone and associated with the hereditary myopathy αB-crystallinopathy. To uncover a molecular mechanism by which the ER-anchored αBC negatively regulates the protein aggregation, we isolated proteins that bind to the ER-anchored αBC and identified the lysosomal protease cathepsin D (CTSD) as one such interacting protein. Proteolytically active CTSD is produced by multi-step processing of pro-cathepsin D (proCTSD), which is initially synthesized in the ER and delivered to lysosomes. When overexpressed, CTSD itself prevented the coexpressed R120G αBC variant from aggregating. This anti-aggregate activity was also elicited upon overexpression of the W383C CTSD variant, which is predominantly sequestered in the ER and consequently remains unprocessed, suggesting that proCTSD, rather than mature CTSD, serves to suppress the aggregation of the R120G αBC variant. Meanwhile, overexpression of the A58V CTSD variant, which is identical to wild-type CTSD except for the Ala58Val substitution within the pro-peptide, did not suppress the protein aggregation, indicating that the integrity of the pro-peptide is required for proCTSD to exert its anti-aggregate activity. Based on our previous finding that overexpression of the ER transmembrane protein CLN6 (ceroid-lipofuscinosis, neuronal 6), identified as an interacting protein of the ER-anchored αBC, prevents the R120G αBC variant from aggregating, the CLN6-proCTSD coupling was hypothesized to underpin the functionality of proCTSD within the ER. Indeed, CTSD, when overexpressed in CLN6-depleted cells, was unable to exert its anti-aggregate activity, supporting our view. Collectively, we show here that proCTSD prevents the protein aggregation through the functional association with CLN6 in the microenvironment surrounding the ER membrane, shedding light on a novel aspect of proCTSD and its potential involvement in CTSD-related disorders characterized by the accumulation of aberrant protein aggregates.
Gen Yasui, Syouichi Katayama, Yukihiko Kubota, Hisashi Takatsuka, Masahiro Ito and Tetsuya Inazu : Zinc finger protein 483 (ZNF483) regulates neuronal differentiation and methyl-CpG-binding protein 2 (MeCP2) intracellular localization., Biochemical and Biophysical Research Communications, Vol.568, 68-75, 2021.
(要約)
Rett syndrome (OMIM #312750) is a developmental neurological disorder that is caused by a mutation in methyl-CpG-binding protein 2 (MeCP2). MeCP2 localizes to the nucleus, binds to methylated DNA, and regulates gene expression during neuronal development. MeCP2 assembles multiple protein complexes and its functions are controlled by interactions with its binding partners. Therefore, functional analysis of MeCP2 binding proteins is important. Previously, we proposed nine MeCP2-binding candidates in the cerebral cortex. In this study, we characterized and examined the function of the MeCP2 binding protein zinc finger protein 483 (ZNF483) to determine the significance of the MeCP2-ZNF483 interaction in neuronal development. Phylogenetic profiling revealed that the ZNF483 protein is broadly conserved in metazoans. In contrast, MeCP2 was obtained during evolution to chordates. To investigate ZNF483 functions, ZNF483-knockout P19 cell lines were established using the CRISPR-Cas9 system. These cell lines showed decreased cell proliferation, altered aggregate formation, decreased neuronal marker NeuN expression, and altered MeCP2 phosphorylation patterns. Notably, cytosolic localization of MeCP2 was enhanced by ZNF483-overexpression. Taken together, we propose that ZNF483 might be involved in the promotion of neuronal differentiation by regulating the subcellular localization of MeCP2 in P19 cells.
Atsushi Morii, Syouichi Katayama and Tetsuya Inazu : Establishment of a Simple Method for Inducing Neuronal Differentiation of P19 EC Cells without Embryoid Body Formation and Analysis of the Role of Histone Deacetylase 8 Activity in This Differentiation., Biological & Pharmaceutical Bulletin, Vol.43, No.7, 1096-1103, 2020.
(要約)
227 medium further enhanced differentiation. Furthermore, cells differentiated by the conventional method, as well as the new method, showed identical expression of the mature neuronal marker, neuronal nuclei. To determine whether our approach could be applied for neuronal studies, we measured histone deacetylase 8 (HDAC8) activity using an HDAC8 inhibitor and HDAC8-knockout P19 EC cells. Inhibition of HDAC8 activity suppressed neuronal maturation. Additionally, HDAC8-knockout cell lines showed immature differentiation compared to the wild-type cell line. These results indicate that HDAC8 directly regulates the neuronal differentiation of P19 EC cells. Thus, our method involving P19 EC cells can be used as an experimental system to study the nervous system. Moreover, this method is suitable for screening drugs that affect the nervous system and cell differentiation.
Muhamad Fahmi, Gen Yasui, Kaito Seki, Syouichi Katayama, Takako Kaneko-Kawano, Tetsuya Inazu, Yukihiko Kubota and Masahiro Ito : In Silico Study of Rett Syndrome Treatment-Related Genes, MECP2, CDKL5, and FOXG1, by Evolutionary Classification and Disordered Region Assessment, International Journal of Molecular Sciences, Vol.20, No.22, 2019.
(要約)
) correspond to distinct neurodevelopmental disorders, given that a series of studies have indicated that RTT is also caused by alterations in either one of these genes. We investigated the evolution and molecular features of MeCP2, CDKL5, and FOXG1 and their binding partners using phylogenetic profiling to gain a better understanding of their similarities. We also predicted the structural order-disorder propensity and assessed the evolutionary rates per site of MeCP2, CDKL5, and FOXG1 to investigate the relationships between disordered structure and other related properties with RTT. Here, we provide insight to the structural characteristics, evolution and interaction landscapes of those three proteins. We also uncovered the disordered structure properties and evolution of those proteins which may provide valuable information for the development of therapeutic strategies of RTT.
Makoto Fukui, Syouichi Katayama, Yukinobu Ikeya and Tetsuya Inazu : Yokukansan, a Kampo medicine, enhances the level of neuronal lineage markers in differentiated P19 embryonic carcinoma cells., Heliyon, Vol.5, No.10, 2019.
(要約)
Yokukansan (YKS), a traditional Japanese Kampo medicine, affects neurological and psychiatric disorders. It ameliorates hippocampal neurogenesis in animals. However, its effect on neuronal cell differentiation remains unclear. Therefore, we investigated the effects of YKS on pluripotent P19 embryonic carcinoma cells as neuronal differentiation model cells. Western blotting and immunocytochemistry revealed that 10 μg/mL YKS treatment during embryoid body formation or neuronal differentiation increased the expression of the neuronal stem cell marker, Nestin, by 1.9-fold and 1.7-fold, respectively, and of the mature neuron marker, NeuN, by 1.5-fold and 1.4-fold, respectively. We examined the effect of YKS on intracellular signaling pathways in P19 cells and found significant elevation in phospho-PDK1 and phospho-mTOR expression (1.1-fold and 1.2-fold, respectively). Therefore, we investigated the effect of PDK1 and mTOR inhibitors on the level of neuronal lineage markers. We found that the mTOR inhibitor significantly abolished the YKS effect on the level of neuronal lineage markers. Moreover, to identify the target(s) of YKS, antibody array analysis that simultaneously detects 16 phosphorylated proteins was performed. YKS significantly upregulated 10 phosphorylated proteins including PDK1, Akt, AMPK, PRAS40, mTOR, p70 S6 kinase, GSK-3α, Bad and ERK1/2 under cell proliferation conditions. These results suggest that YKS simultaneously activates multiple signaling pathways. Thus, we concluded that YKS enhances the level of neuronal lineage markers in differentiated P19 cells, however it does not induce neuronal differentiation. Furthermore, mTOR is the predominant mediator of the YKS effect on these cells.
Syouichi Katayama and Tetsuya Inazu : Straightforward and rapid method for detection of cyclin-dependent kinase-like 5 activity., Analytical Biochemistry: Methods in the Biological Sciences, Vol.566, 58-61, 2018.
(要約)
Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase, with its gene mutation leading to a neurodevelopmental disorder. Pathogenic point mutations are mostly observed within the catalytic domain of CDKL5, therefore loss of catalytic activity may be related to disease onset. However, this hypothesis has rarely been demonstrated. Here, we report an efficient method for detecting CDKL5 activity. Appropriately, CDKL5 underwent autophosphorylation following expression in Escherichia coli, with autophosphorylated CDKL5 detected as a band shift by phos-tag SDS-PAGE, without enzyme purification. Thus, this protocol is useful for examining the relationship between disease-causing mutations and their activity.
Syouichi Katayama, Atsushi Morii, O Juliet Makanga, Takayoshi Suzuki, Naoki Miyata and Tetsuya Inazu : HDAC8 regulates neural differentiation through embryoid body formation in P19 cells., Biochemical and Biophysical Research Communications, Vol.498, No.1, 45-51, 2018.
(要約)
Histone acetylation and deacetylation correlate with diverse biological phenomena through gene transcription. Histone deacetylases (HDACs) regulate deacetylation of histones and other proteins. However, as a member of the HDAC family, HDAC8 function during neurodevelopment is currently unknown. Therefore, we investigated HDAC8 function during neurodevelopment by examining embryoid body (EB) formation in P19 cells. HDAC8-selective inhibitor (NCC-149) (HDAC8i)-treated cells showed smaller EBs than non-treated cells, as well as reduced expression levels of the neuronal marker, NeuN. Additionally, HDAC8i treatment led to inhibition of cellular proliferation by G2/M phase accumulation and downregulated cyclin A2 and cyclin B1 gene expression. Furthermore, two independent HDAC8 knockout cell lines were established by CRISPR-Cas9, which resulted in smaller EBs, similar to HDAC8i-treated cells. These results suggest that HDAC8 regulates neural differentiation by exerting control of EB formation.
Ami Oi, Syouichi Katayama, Naoya Hatano, Yasunori Sugiyama, Isamu Kameshita and Noriyuki Sueyoshi : Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)., Biochemical and Biophysical Research Communications, Vol.482, No.2, 239-245, 2016.
(要約)
Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization.
Yasunori Sugiyama, Sho Yamashita, Yuuki Uezato, Yukako Senga, Syouichi Katayama, Naoki Goshima, Yasushi Shigeri, Noriyuki Sueyoshi and Isamu Kameshita : Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay., Analytical Biochemistry: Methods in the Biological Sciences, Vol.513, 47-53, 2016.
(要約)
To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.
Antonius Christianto, Syouichi Katayama, Isamu Kameshita and Tetsuya Inazu : A novel CDKL5 mutation in a Japanese patient with atypical Rett syndrome., Clinica Chimica Acta, Vol.459, 132-136, 2016.
(要約)
Rett syndrome (RTT) is a severe X-linked dominant inheritance disorder with a wide spectrum of clinical manifestations. Mutations in Methyl CpG binding protein 2 (MECP2), Cyclin dependent kinase-like 5 (CDKL5) and Forkhead box G1 (FOXG1) have been associated with classic and/or variant RTT. This study was conducted to identify the responsible gene(s) in atypical RTT patient, and to examine the effect of the mutation on protein function. DNA sequence analysis showed a novel heterozygous mutation in CDKL5 identified as c.530A>G which resulted in an amino acid substitution at position 177, from tyrosine to cysteine. Genotyping analysis indicated that the mutation was not merely a single nucleotide polymorphism (SNP). We also revealed that patient's blood lymphocytes had random X-chromosome inactivation (XCI) pattern. Further examination by bioinformatics analysis demonstrated the mutation caused damage or deleterious in its protein. In addition, we demonstrated in vitro kinase assay of mutant protein showed impairment of its activity. Taken together, the results suggested the mutant CDKL5 was responsible for the disease.
(キーワード)
Female / Humans / Japan / Middle Aged / Mutation / Protein-Serine-Threonine Kinases / Rett Syndrome
Yukako Senga, Kazutoshi Akizuki, Syouichi Katayama, Yasushi Shigeri, Isamu Kameshita, Atsuhiko Ishida and Noriyuki Sueyoshi : High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli., Biochemical and Biophysical Research Communications, Vol.475, No.3, 277-282, 2016.
(要約)
We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.
Syouichi Katayama, Yukako Senga, Ami Oi, Yosuke Miki, Yasunori Sugiyama, Noriyuki Sueyoshi and Isamu Kameshita : Expression analyses of splice variants of zebrafish cyclin-dependent kinase-like 5 and its substrate, amphiphysin 1., Gene, Vol.583, No.1, 15-23, 2016.
(要約)
Mammalian cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase mainly expressed in the central nervous system and believed to be involved in neuronal functions. However, the functions of CDKL5 in fishes have not been investigated. Therefore, in this study, we cloned and characterized zebrafish CDKL5 (zCDKL5) and its substrate, amphiphysin 1 (zAmph1). Two alternative splice variants of zCDKL5, zCDKL5-Long (zCDKL5-L) and zCDKL5-Short (zCDKL5-S), and three splice variants of zAmph1, zAmph1a, zAmph1b and zAmph1c, were cloned from a zebrafish cDNA library. Using zAmph1a point mutants, we identified Ser-285 and Ser-293 as phosphorylation sites of zAmph1a by CDKL5. Transiently expressed zCDKL5-L and zCDKL5-S colocalized with zAmph1a in the cytoplasm of 293T cells. RT-PCR analysis revealed that zCDKL5-L was first observed 12hours post-fertilization (hpf) and increased thereafter, while zCDKL5-S appeared just after fertilization. zAmph1a was detected in all embryogenic stages and zAmph1b appeared from 12hpf, but the expression of zAmph1c was not observed in our experiments. In adult fish, zCDKL5-L was mainly expressed in the brain, but zCDKL5-S showed ubiquitous expression. zAmph1a was observed most abundantly in the eyes, whereas zAmph1b was predominantly expressed in the brain. zAmph1c was scarcely detected. These results suggest that phosphorylation of Amph1 by CDKL5 may be a common feature throughout animal species.
Yasunori Sugiyama, Syouichi Katayama, Isamu Kameshita, Keiko Morisawa, Takuma Higuchi, Hiroshi Todaka, Eiji Kinoshita, Emiko Kinoshita-Kikuta, Tohru Koike, Taketoshi Taniguchi and Shuji Sakamoto : Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE., MethodsX, Vol.2, 469-474, 2015.
(要約)
Protein kinase expression and activity play important roles in diverse cellular functions through regulation of phosphorylation signaling. The most commonly used tools for detecting the protein kinase are protein kinase-specific antibodies, and phosphorylation site-specific antibodies were used for detecting activated protein kinase. Using these antibodies, only one kinase was analyzed at a time, however, a method for analyzing the expression and activation of a panel of protein kinases in cells is not established. Therefore, we developed a combined method using Multi-PK antibody and Phos-tag SDS-PAGE for profiling the expression and phosphorylation state of intracellular protein kinases. Using the new method, changes in the expression and phosphorylation state of various protein kinases were detected in cells treated with anticancer agent which inhibit multiple tyrosine kinase activities. Therefore, the new method is a useful technique for analysis of intracellular protein kinases.•Multi-PK antibody recognizes a wide variety of protein kinases in various species.•Using Phos-tag SDS-PAGE, phosphorylated proteins are visualized as slower migration bands compared with corresponding non-phosphorylated proteins.•This combined method can be used for detecting changes in the expression and phosphorylation state of various intracellular protein kinases.
Syouichi Katayama, Noriyuki Sueyoshi and Isamu Kameshita : Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5)., Biochemistry, Vol.54, No.19, 2975-2987, 2015.
(要約)
Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase known to be associated with X-linked neurodevelopmental disorders. In a previous study, we identified amphiphysin 1 (Amph1) as a potential substrate for CDKL5 and identified a single phosphorylation site at Ser-293. In this study, we investigated the molecular mechanisms of substrate recognition by CDKL5 using Amph1 as a model substrate. Amph1 served as an efficient CDKL5 substrate, whereas Amph2, a structurally related homologue of Amph1, was not phosphorylated by CDKL5. The sequence around the Amph1 phosphorylation site is RPR(293)SPSQ, while the corresponding sequence in Amph2 is IPK(332)SPSQ. To define the amino acid sequence specificity of the substrate, various point mutants of Amph1 and Amph2 were prepared and phosphorylated by CDKL5. Both Amph2(I329R) and Amph1 served as efficient CDKL5 substrates, but Amph1(R290I) did not, indicating that the arginyl residue at the P -3 position is critical for substrate recognition. With regard to prolyl residues around the phosphorylation site of Amph1, Pro-291 at the P -2 position, but not Pro-294 at the P +1 position, is indispensable for phosphorylation by CDKL5. Phosphorylation experiments using various deletion mutants of Amph1 revealed that the proline-rich domain (PRD) (amino acids 247-315) alone was not phosphorylated by CDKL5. In contrast, Amph1(247-385), which comprised the PRD and CLAP domains, served as an efficient CDKL5 substrate. These results, taken together, suggest that both the phosphorylation site sequence (RPXSX) and the CLAP domain structure in Amph1 play crucial roles in recognition and phosphorylation by CDKL5.
Isamu Kameshita, Sho Yamashita, Syouichi Katayama, Yukako Senga and Noriyuki Sueyoshi : TandeMBP: generation of a unique protein substrate for protein kinase assays., The Journal of Biochemistry, Vol.156, No.3, 147-154, 2014.
(要約)
Myelin basic protein (MBP) is one of the major components of central nervous system myelin and has multiple sites for protein phosphorylation. Therefore, it has been widely used as a substrate for in vitro assays of various protein kinases. In this study, to obtain more efficient substrates for protein kinase assays than commercially available MBP from bovine brain, we produced various recombinant MBPs using Escherichia coli expression systems. Three splice isoforms of mouse MBP were expressed in E. coli and successfully purified using a new protocol consisting of HCl extraction, urea treatment and affinity purification with HiTrap Chelating HP column. The recombinant MBP isoforms thus obtained served as more efficient substrates for protein kinases than MBP isolated from bovine brain. To generate an even better substrate for protein kinase assays, we produced a hybrid protein composed of two different MBP isoforms connected in tandem, designated TandeMBP. TandeMBP was readily expressed in E. coli and could be purified by the newly developed simple procedure. TandeMBP was phosphorylated by various Ser/Thr protein kinases more efficiently than the other MBP isoforms. Taken together, TandeMBP will become a powerful tool for in vitro assays to analyse various protein kinase activities.
(キーワード)
Amino Acid Sequence / Animals / Blotting, Western / Cattle / Enzyme Assays / Molecular Sequence Data / Myelin Basic Protein / Phosphorylation / Protein Isoforms / Protein Kinases / Recombinant Fusion Proteins / Reproducibility of Results / Sequence Homology, Amino Acid / Substrate Specificity
Mari Sekiguchi, Syouichi Katayama, Naoya Hatano, Yasushi Shigeri, Noriyuki Sueyoshi and Isamu Kameshita : Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder., Archives of Biochemistry and Biophysics, Vol.535, No.2, 257-267, 2013.
(要約)
Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development.
(キーワード)
Amino Acid Sequence / Animals / Brain / Isoelectric Focusing / Male / Mice / Molecular Sequence Data / Mutation / Nerve Tissue Proteins / Phosphorylation / Protein Binding / Protein-Serine-Threonine Kinases / Rett Syndrome / Serine / Tissue Extracts
Syouichi Katayama, Yasunori Sugiyama, Naoya Hatano, Toru Terachi, Noriyuki Sueyoshi and Isamu Kameshita : PKL01, an Ndr kinase homologue in plant, shows tyrosine kinase activity., The Journal of Biochemistry, Vol.152, No.4, 347-353, 2012.
(要約)
Protein phosphorylation by protein tyrosine (Tyr) kinases plays important roles in a variety of signalling pathways in cell growth, differentiation and oncogenesis in animals. Despite the absence of classical Tyr kinases in plants, a similar ratio of phosphotyrosine residues in phosphorylated proteins was found in Arabidopsis thaliana as in human. However, protein kinases responsible for tyrosine phosphorylation in plants except some dedicated dual-specificity kinases still remain unclear. In this study, we found that PKL01, a nuclear Dbf2-related (Ndr) kinase homologue in Lotus japonicus, was autophosphorylated at a tyrosine residue when it was expressed in Escherichia coli, but kinase-dead mutant of PKL01 was not. Tyrosine phophorylation site in PKL01 was identified as Tyr-56 by LC-MS/MS analysis. Recombinant PKL01, which had been dephosphorylated by an alkaline phosphatase, could be phosphorylated again at the Tyr residue when it was incubated with ATP. Furthermore, other Ndr kinases in plants and PKL01 phosphorylated on Tyr residues in the exogenous substrates such as poly(Glu, Tyr)(4:1) and casein. Therefore, the Ndr kinases in plants, which had been assumed as protein serine (Ser)/threonine (Thr) kinases, turned out to be dual-specificity kinases responsible for phosphorylation of Tyr residues and Ser/Thr residues in plant proteins.
Syouichi Katayama : Establishment a straightforward method for detecting catalytic activity of CDKL5 using phos-tag SDS-PAGE, Current Topics in Basic and Translational Research for Development of Innovative Drugs (Dongguk University), Dec. 2021.