A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody

Matsuo, Ritsuko; Ochiai, Wataru; Nakashima, Kinichi; Taga, Tetsuya

doi:10.1016/s0022-1759(00)00313-6

Cited by 68 publications

(54 citation statements)

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“…Recently, STAT3 (signal transduction and activators of transcription 3) and FKHR were identified as substrates of the DYRK1A family. 21,24,40 FKHR is also a target for the PKB/AKT signaling pathway initiated by growth factor receptor activation. [41][42][43][44] The PKB/AKT phosphorylates FKHR at 3 residues (Thr 24 , Ser 256 and Ser 319 ) by phosphoinositide 3-kinase (PI 3 K) pathway that results in the nuclear exit and inactivation of apoptosis related gene.…”

Section: Discussionmentioning

confidence: 99%

“…cytoplasm leads to the suppression of Fas ligand, Bcl-6 and BAD. 21,24,25 In this study, BAD were measured by Northern and Western blot analyses and levels of BAD were compared among cell groups. As shown in Figure 7, cell immortalization greatly reduced the BAD content when compared with the primary keratinocytes (FK).…”

Section: Rnai Ablation Of Dyrk1a Increases Apoptosis Fkhr Translocatmentioning

confidence: 99%

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Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

Chang

Lin

Yang

et al. 2007

Intl Journal of Cancer

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Immortalization is a critical event in virus-related oncogenesis. No enough information, however, is currently available to elucidate the changes that occur in cellular molecules during immortalization. To identify potential cellular markers or regulators involving in immortalization, a paired-cell model of primary foreskin keratinocytes (FK) and HPV16 immortalized foreskin keratinocytes were established. Using mRNA differential display, RT-PCR and Northern blot methods, we have identified and confirmed that Dyrk1a (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) is present and increased in HPV16 immortalized cells, but is absent in primary keratinocytes. Moreover, transfection of E7 siRNA oligo into immortalized cells leads to a diminishing E7 expression and the eventual disappearance of Dyrk1a. Similar results of Dyrk1a expressional differences could also be seen when tissue specimens were compared using LCM/real-time PCR and immunohistochemistry analysis; malignant cervical lesions contain significantly more DYRK1A than normal tissue. It was also demonstrated that raised DYRK1A could rearrange the cellular localization of FKHR (forkhead in rhabdomyosarcoma), an apoptosis activator, and suppress BAD. Importantly, this phenomenon can be reversed when endogenous Dyrk1a was knocked down in immortalized cells by RNA interference. These results suggest that the raised Dyrk1a in HPV16 immortalized keratinocytes and cervical lesions may serve as a candidate antiapoptotic factor in the FKHR regulated pathway and initiate immortalization and tumorigenesis gradually. ' 2007 Wiley-Liss, Inc.Key words: HPV16; keratinocytes; Dyrk1a; immortalization; apoptosis Mammalian cells have a limited life span and proliferating capacity when cultivated in vitro. Replicate senescence or the terminal arrest state, has been found to be accompanied by several molecular changes that lead to an activated suppression of the cell cycle and the shortening of the telomeres. 1 Similar to apoptosis, cellular senescence is thought to be a mechanism of tumor suppression because it prevents the outgrowth of cells that have acquired mutations in genes rendering them cancerous. Consistent with this model, several tumor suppressor genes (such as, overexpressed p16 or ZNF217, activated p53 and deregulated Rassf1a or c-myc, etc) or their products have been reported and shown to be associated in parallel with telomere irregularities at the onset of cellular senescence. 2 Telomere shortening has been shown to generate unstable DNA leading to p53 activation and the subsequent transcriptional induction of the cdk inhibitor p21 CIP13 that moves cells into senescence state. Therefore, preventing telomere erosion by ectopic expression of telomerase is sufficient to immortalized primary cells in vitro and thus extend life span. [4][5][6] Although it works in human fibroblast, telemorase alone, however, is not sufficient to induce immortalization in human keratinocytes and mammary epithelial cells. 7 To identify other factors that are involved ...

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Section: Discussionmentioning

confidence: 99%

Section: Rnai Ablation Of Dyrk1a Increases Apoptosis Fkhr Translocatmentioning

confidence: 99%

Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

Chang

Lin

Yang

et al. 2007

Intl Journal of Cancer

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“…To identify kinases that phosphorylate c-Abl at Thr735, we performed expression-cloning analysis by using antiphospho-c-Abl(Thr735) antibody (T735-Ab) ( Figure 1a) (Matsuo et al, 2001). The LE392 strain of Escherichia coli transformed with GST-c-Abl(683-780) wild-type (wt) or the GST-c-Abl(683-780) T735A mutant, in which Thr735 is substituted with Ala, was infected with phage expression libraries prepared from the human fetal brain.…”

Section: Identification Of Thr735 Kinases By Expression Cloningmentioning

confidence: 99%

“…Identification of Thr735 kinases using phosphospecific antibodies was performed as described previously (Matsuo et al, 2001;Taira et al, 2007), with minor modification. Briefly, LE392 strain of E. coli were transfected with GST-c-Abl(683-790) wild type or T735A by electroporation.…”

Section: Screening Of the Kinases That Phosphorylate C-abl At Thr735mentioning

confidence: 99%

TTK/Mps1 controls nuclear targeting of c-Abl by 14-3-3-coupled phosphorylation in response to oxidative stress

et al. 2008

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Upon exposure to genotoxic stress, the c-Abl tyrosine kinase is released from cytoplasmic 14-3-3 proteins and then is targeted to the nucleus. Phosphorylation of Thr735 in c-Abl is critical for binding to 14-3-3; however, kinases responsible for this phosphorylation are unknown. Here, we identify CLK1, CLK4, MST1, MST2 and TTK (also known as Mps1) as novel Thr735 kinases in vitro by expression cloning strategy using phosphospecific antibody. We also demonstrate that ectopic expression of these kinases is capable for phosphorylation of Thr735 in cells. Importantly, upon exposure to oxidative stress, phosphorylation of Thr735 is transiently upregulated, and the status of this phosphorylation remains unchanged in cells silenced for CLK1, CLK4, MST1 or MST2. By contrast, knockdown of TTK attenuates phosphorylation of Thr735, suggesting that TTK is a physiological kinase that phosphorylates Thr735. In concert with these results, we show that, in cells silenced for TTK, c-Abl is accumulated in the nucleus even in unstressed condition and no further targeting into the nucleus occurs after oxidative stress. Moreover, nuclear entrapment of c-Abl by knocking down TTK enhances oxidative stress-induced apoptosis. These findings provide evidence that TTK phosphorylates c-Abl at Thr735 and that this phosphorylation is of importance to the cytoplasmic sequestration of c-Abl.

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“…Although the biological function of DYRK1A is not known, DYRK1A interacts in vivo with several transcription factors. Additionally, DYRK1A phosphorylates several substrates in vitro, This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04 -12-1085) on May 25, 2005. including the signal transducer and activator of transcription 3 (Matsuo et al, 2001), the eukaryotic initiation factor 2B⑀ (Woods et al, 2001a), the microtubule-associated protein Tau (Woods et al, 2001a), the transcription factor of the Forkhead family (FHKR) (Woods et al, 2001b), dynamin (Chen-Hwang et al, 2002), glycogen synthase (Skurat and Dietrich, 2004), cyclin L2 (De Graaf et al, 2004), and 14-3-3 (Kim et al, 2004), indicating that DYRK1A may participate in several biochemicals pathways (reviewed in Galceran et al, 2003;Hämmerle et al, 2003b). In vivo, DYRK1A interacts with and activates Gli-1-dependent gene transcription (Mao et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

DYRK1A Enhances the Mitogen-activated Protein Kinase Cascade in PC12 Cells by Forming a Complex with Ras, B-Raf, and MEK1

Kelly

Rahmani

2005

MBoC

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Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) is the human homologue of the Drosophila mnb (minibrain) gene. In Drosophila, mnb is involved in postembryonic neurogenesis. In human, DYRK1A maps within the Down syndrome critical region of chromosome 21 and is overexpressed in Down syndrome embryonic brain. Despite its potential involvement in the neurobiological alterations observed in Down syndrome patients, the biological functions of the serine/threonine kinase DYRK1A have not been identified yet. Here, we report that DYRK1A overexpression potentiates nerve growth factor (NGF)-mediated PC12 neuronal differentiation by up-regulating the Ras/MAP kinase signaling pathway independently of its kinase activity. Furthermore, we show that DYRK1A prolongs the kinetics of ERK activation by interacting with Ras, B-Raf, and MEK1 to facilitate the formation of a Ras/B-Raf/MEK1 multiprotein complex. These data indicate that DYRK1A may play a critical role in Ras-dependent transducing signals that are required for promoting or maintaining neuronal differentiation and suggest that overexpression of DYRK1A may contribute to the neurological abnormalities observed in Down syndrome patients. INTRODUCTIONMinibrain (mnb)/dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) gene is a member of a growing family of protein kinases called DYRK. In Drosophila, mnb seems to play an essential role during postembryonic neurogenesis. Mutant flies are characterized by a marked reduction in size of the adult optic lobes and the central brain hemispheres. This is caused by the abnormal spacing of neuroblasts and a reduction in the production of neuronal progeny (Tejedor et al., 1995). At least seven closely related homologous mammalian kinases in the DYRK family have since been isolated (Guimera et al., 1996(Guimera et al., , 1999Shindoh et al., 1996;Song et al., 1996Song et al., , 1997Raich et al., 2003). The DYRK family possesses serine and threonine phosphorylation activity as well as autophosphorylation activity on tyrosine residues (Kentrup et al., 1996;Becker et al., 1998;Becker and Joost, 1999;Himpel et al., 2000). Their kinase activity is dependent on the YXY motif in the activation loop of the catalytic domain, which is located at the same position as the characteristic TXY motif of the mitogen-activated protein kinases (MAPKs), suggesting an activation mechanism similar to that of the MAPK (Himpel et al., 2000). Closely related enzymes in lower eukaryotes also have been isolated, including Yak1p in Saccharomyces cerevisiae (Garrett and Broach, 1989), Pom1p in Schizosaccharomyces pombe (Bahler and Pringle, 1998), and YakA in Dictyostelium discoideum (Van Es et al., 2001). Although not much is known about their cellular functions, they all seem to be involved in the regulation of the cell growth and/or development.In the DYRK family, DYRK1A has been the best characterized to date. The human Dyrk1A gene maps to the 21q22.2 region of chromosome 21, in the Down syndrome critical region (Rah...

show abstract

A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody

Cited by 68 publications

References 30 publications

Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

Increased expression of Dyrk1a in HPV16 immortalized Keratinocytes enable evasion of apoptosis

TTK/Mps1 controls nuclear targeting of c-Abl by 14-3-3-coupled phosphorylation in response to oxidative stress

DYRK1A Enhances the Mitogen-activated Protein Kinase Cascade in PC12 Cells by Forming a Complex with Ras, B-Raf, and MEK1

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