Identification of kinase substrates by bimolecular complementation assays

Pusch, Stefan; Harashima, Hirofumi; Schnittger, Arp

doi:10.1111/j.1365-313x.2011.04862.x

Cited by 27 publications

(27 citation statements)

References 47 publications

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“…However, we failed to detect the in vivo CRY2-CK1.3 or CRY2-CK1.4 interactions by coimmunoprecipitation after numerous attempts, which may be due to the transient and weak interaction between CRY2 and CK1.3 or CK1.4. Indeed, similar phenomena (weak interaction between kinases and substrates) were reported in other studies on kinases, confirming the transient "kiss-and-run" nature of the enzyme-substrate interactions in vivo (Gudesblat et al, 2012;Pusch et al, 2012).…”

Section: Arabidopsis Ck13 and Ck14 Encode Functional Ck1ssupporting

confidence: 85%

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling

et al. 2013

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ORCID IDs: 0000-0002-0471-8285 (S-T.T.); 0000-0002-4853-8278 (C.D.).Casein kinase1 (CK1) plays crucial roles in regulating growth and development via phosphorylating various substrates throughout the eukaryote kingdom. Blue light is crucial for normal growth of both plants and animals, and blue light receptor cryptochrome2 (CRY2) undergoes blue light-dependent phosphorylation and degradation in planta. To study the function of plant CK1s, systematic genetic analysis showed that deficiency of two paralogous Arabidopsis thaliana CK1s, CK1.3 and CK1.4, caused shortened hypocotyls, especially under blue light, while overexpression of either CK1.3 or CK1.4 resulted in the insensitive response to blue light and delayed flowering under long-day conditions. CK1.3 or CK1.4 act dependently on CRY2, and overexpression of CK1.3 or CK1.4 significantly suppresses the hypersensitive response to blue light by CRY2 overexpression. Biochemical studies showed that CK1.3 and CK1.4 directly phosphorylate CRY2 at Ser-587 and Thr-603 in vitro and negatively regulate CRY2 stability in planta, which are stimulated by blue light, further confirming the crucial roles of CK1.3 and CK1.4 in blue light responses through phosphorylating CRY2. Interestingly, expression of CK1.3 and CK1.4 is stimulated by blue light and feedback regulated by CRY2-mediated signaling. These results provide direct evidence for CRY2 phosphorylation and informative clues on the mechanisms of CRY2-mediated light responses.

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Section: Arabidopsis Ck13 and Ck14 Encode Functional Ck1ssupporting

confidence: 85%

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling

et al. 2013

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“…In addition, there could also be other plant‐specific phospho‐targets of CDKB1 during HR. The few targets of CDK action identified in Arabidopsis so far suggest a rather large number of species‐specific CDK targets (Pusch et al , 2012). Hence, an unbiased forward approach to identify additional CDK targets under DNA damage conditions might be powerful to further understand how HR is controlled by CDKs.…”

Section: Discussionmentioning

confidence: 99%

The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis

et al. 2016

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Upon DNA damage, cyclin‐dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology‐dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant‐specific B1‐type CDKs (CDKB1s) and the class of B1‐type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1‐CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA. CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell‐cycle activity is blocked after DNA damage, CDKB1‐CYCB1 complexes are specifically activated to mediate HR.

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“…Degradation of URA3 prevents the conversion of 5-fluoroorotic acid monohydrate (5-FOA) to the toxic compound 5-fluorouracil; thus, when there is a PPI, the cells survive in the presence of 5-FOA (64). Such an assay was used to detect substrates of CDKA in Arabidopsis (65). A variation of the SUS is the bimolecular fluorescence complementation [BiFC; also called split yellow fluorescent protein (YFP)] assay (66).…”

Section: Arrangement Of Detected Motif In Detected Substratementioning

confidence: 99%

“…Pusch and collaborators used both the SUS and BiFC systems to detect and validate in vivo substrates for the Arabidopsis CDKA. A few of the substrates were further validated to show that CDKA phosphorylates proteins that control the redox state by the cell cycle (65).…”

Section: Arrangement Of Detected Motif In Detected Substratementioning

confidence: 99%

Revisiting protein kinase–substrate interactions: Toward therapeutic development

et al. 2016

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Despite the efforts of pharmaceutical companies to develop specific kinase modulators, few drugs targeting kinases have been completely successful in the clinic. This is primarily due to the conserved nature of kinases, especially in the catalytic domains. Consequently, many currently available inhibitors lack sufficient selectivity for effective clinical application. Kinases phosphorylate their substrates to modulate their activity. One of the important steps in the catalytic reaction of protein phosphorylation is the correct positioning of the target residue within the catalytic site. This positioning is mediated by several regions in the substrate binding site, which is typically a shallow crevice that has critical subpockets that anchor and orient the substrate. The structural characterization of this protein-protein interaction can aid in the elucidation of the roles of distinct kinases in different cellular processes, the identification of substrates, and the development of specific inhibitors. Because the region of the substrate that is recognized by the kinase can be part of a linear consensus motif or a nonlinear motif, advances in technology beyond simple linear sequence scanning for consensus motifs were needed. Cost-effective bioinformatics tools are already frequently used to predict kinase-substrate interactions for linear consensus motifs, and new tools based on the structural data of these interactions improve the accuracy of these predictions and enable the identification of phosphorylation sites within nonlinear motifs. In this Review, we revisit kinase-substrate interactions and discuss the various approaches that can be used to identify them and analyze their binding structures for targeted drug development. The Catalytic Domain of Eukaryotic Protein KinasesTypically, eukaryotic protein kinases are composed of nonconserved regulatory domains and a conserved catalytic core of~250 amino acid residues that binds and anchors substrates and is responsible for catalysis (1). The catalytic domain consists of two lobes called N and C (also known as small and large lobes, respectively), named for their N-or C-terminal position, respectively, within the domain. The N-lobe consists of five-stranded, anti-parallel b sheets that are an essential part of the adenosine triphosphate (ATP) binding site, whereas the C-lobe is mostly helical (Fig. 1A). The active-site cleft, which contains the ATP binding site, lies between the two lobes (2). In an activated kinase, the lobes converge to form a deep cleft where the adenine ring of ATP binds such that the g-phosphate is positioned at the outer edge where the transfer of the phosphoryl group occurs, whereas the adenosine moiety is buried in a hydrophobic region of the pocket (Fig. 1B). Adjacent to the ATP binding pocket is a shallow crevice called the substrate binding site (SBS) that anchors the substrate and correctly positions the phosphorylatable residue (2).Catalysis is mediated by opening and closing of this active-site cleft. Substrates are anchored and p...

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Identification of kinase substrates by bimolecular complementation assays

Cited by 27 publications

References 47 publications

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling

The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis

Revisiting protein kinase–substrate interactions: Toward therapeutic development

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