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  • It has been shown that CK phosphorylates

    2019-10-31

    It has been shown that CK1δ phosphorylates α-, β- and γ-tubulin in vitro and that CK1δ specifically interacts with the trans Golgi network, COPI positive vesicles, and centrosomes in interphase cells [11], [12], [13], [14]. Moreover CK1δ is also associated with granular particles that are associated with microtubules [11], [15], and recently, it was shown that siRNA induced knockdown of CK1δ inhibits microtubule nucleation at the Golgi apparatus [16]. CK1δ is recruited to the spindle apparatus during mitosis [17] and its association is significantly enhanced upon DNA-damage induced by camptothecin, etoposide or γ-irradiation [15]. CK1δ inhibition by specific small molecule inhibitors led to structural alterations of centrosomes, the formation of multipolar spindles, and inhibition of mitosis [17], [18]. Furthermore, at lower concentrations of CK1 specific inhibitors, mitotic spindle dynamics was affected leading to WZ4003 arrest and, depending on the cellular background, to apoptosis in a dose-dependent manner [11], [19]. In order to maintain genomic stability under conditions of genotoxic stress, CK1δ regulates microtubule and spindle dynamics by phosphorylation of Sid4 thereby delaying cytokinesis [20] as well as by phosphorylation of MAP4, MAP1A, tau, and Stathmin [4], [10], [15], [21]. In summary, the association of CK1δ to α-tubulin and various proteins involved in mitosis points to a role of CK1δ in regulating key aspects of cell division and mitotic progression and by performing Michaelis–Menten kinetics we identified α-tubulin as the preferred interaction partner of CK1 isoforms [11]. Since CK1 isoforms contribute to the pathogenesis of proliferative diseases such as cancer, the development of CK1-specific small molecule inhibitors draws more and more attention (reviewed in Ref. 4). However, lack of stringent specificity of kinase inhibitors is still a challenging problem [22] and indeed a variety of pretended “specific” CK1 inhibitors are known to have off-target effects [23] causing severe side effects [24].
    Material and methods
    Results CK1 is linked to the regulation of various cellular functions being involved in the coordination of cell cycle progression, spindle dynamics, and chromosome segregation (reviewed in Ref. 4). CK1δ displays a high affinity to the spindle apparatus upon DNA-damage and is involved in the regulation of mitotic progression [11], [15], [17], [18], [31]. Since CK1δ interacts with and phosphorylates α/β-tubulin, the characterisation of the protein–protein interface between CK1δ and α/β-tubulin would allow identification of functional motifs that could be used to perturb the interaction between CK1δ and tubulin. For the assessment of binding sites a library of peptides covering the CK1δ amino acid sequence was synthesised. Since we could show in previous studies that CK1 isoforms display a significantly higher affinity toward α-tubulin than toward β-tubulin [11], we concentrated on the mapping of the CK1δ/α-tubulin interface.
    Discussion It has been shown that CK1 is linked to various cellular functions being involved in cell cycle progression and spindle dynamics (reviewed in Ref. 4). Especially, CK1δ is tightly associated with microtubules and known to phosphorylate several tubulin isoforms including α-tubulin [11], [15], [17], [18]. In our studies we shed light on the protein–protein interface between CK1δ and α-tubulin to identify functional motifs that could be used as novel biological tool to perturb mitosis in proliferative diseases.
    Funding Financial support by AIRC (grant IG14180) to LAP is gratefully acknowledged. Work in the lab of Uwe Knippschild is funded by the Deutsche Forschungsgemeinschaft (DFG) (KN356/6-1).
    Conflict of interest
    Acknowledgements
    Introduction The p53 tumour suppressor protein is a latent transcription factor which is activated by a wide range of cellular stresses (for review, see [1], [2]). Activation of p53 in response to DNA damage involves the release of p53 from complex formation with its negative regulatory partner, MDM2, and is accompanied by a sequential series of phosphorylation and acetylation events which include phosphorylation of serines 15, 20 and 33 at the N-terminus of the protein (for review, see [3]). The initial modification, phosphorylation of serine 15, is mediated by the ATM protein kinase in vivo [4], [5]. (Serine 15 and serine 37 can also be modified in vitro by the DNA-activated protein kinase (DNA-PK) [6].) Serine 15 phosphorylation stimulates p53-dependent transactivation through increased interaction with p300/CBP [7], [8] and may contribute to disruption of the p53-MDM2 association [9], [10]. Serine 15 modification may also nucleate subsequent modifications such as the acetylation of the C-terminus through increased binding of p300/CBP [11]. Recent evidence has also established that serine 20, a key phosphorylation site which is modified following DNA damage [12], plays a critical role in the disassociation of p53 and MDM2. Similarly, threonine 18 is phosphorylated in vivo and can also contribute to the dissociation of the p53-MDM2 complex [13], [14]. Moreover, extensive threonine 18 and serine 20 phosphorylation has been detected in a panel of human breast cancers with wild-type (WT) p53 status [14] underscoring their biomedical relevance. To date, however, the protein kinases which phosphorylate threonine 18 and serine 20 have not been established.