High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer

Bhoir, Siddhant; Shaik, Althaf; Thiruvenkatam, Vijay; Kirubakaran, Sivapriya

doi:10.1038/s41598-018-22744-5

Cited by 16 publications

(31 citation statements)

References 44 publications

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“…A few PTH anti-psychotics were identified in a compounds library screen as good inhibitors of TLK1 ( Ronald et al., 2013 ). To find additional inhibitors, recombinant TLK1B was purified as previously described ( Bhoir et al., 2018 ) ( Figure 1 A). The enzymatic activity was determined using the ADP-Hunter reagents and a Nek1 peptide containing the T141 target site.…”

Section: Resultsmentioning

confidence: 99%

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

et al. 2020

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Summary Through in vitro kinase assays and docking studies, we report the synthesis and biological evaluation of a phenothiazine analog J54 with potent TLK1 inhibitory activity for prostate cancer (PCa) therapy. Most PCa deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa. Treatments that can suppress the conversion to mCRPC have high potential to be rapidly implemented in the clinics. ADT results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DNA damage response that typically results in temporary cell-cycle arrest of androgen-responsive PCa cells, whereas its abrogation leads to apoptosis. We studied J54 as a potent inhibitor of this axis and as a mediator of apoptosis in vitro and in LNCaP xenografts, which has potential for clinical investigation in combination with ADT. J54 has low affinity for the dopamine receptor in modeling and competition studies and weak detrimental behavioral effects in mice and C. elegans.

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Section: Resultsmentioning

confidence: 99%

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

et al. 2020

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“…Kinase-generated ADP is converted back to ATP which is then driving a luciferase reaction and quantified by luminescence. This method has, for example, been employed for the characterization of recombinant ribosomal S6 kinase 2 (RSK2) and Tousled-like kinase 1B (Bhoir, Shaik, Thiruvenkatam, & Kirubakaran, 2018;Utepbergenov et al, 2016). In addition, there exist several variants of non-antibody FRET assays (Liu et al, 2010;Lawrence & Wang, 2007;Newman & Josiah, 2004).…”

Section: In-vitro Kinase Activity Assaysmentioning

confidence: 99%

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

Galicia

Aldehaiman

Hong

et al. 2019

Methods in Enzymology

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Protein tyrosine kinases (PTKs) are key signaling molecules and important drug targets. Although the efficient recombinant production of active PTKs is important for both pharmaceutical industry and academic research, most PTKs are still obtained from conventional, expensive and time-consuming insect-cell based expression. Host toxicity, kinase inactivity, insolubility and heterogeneity are among the reasons thought to preclude PTK expression in Escherichia coli. Herein we review these presumed roadblocks and their possible solutions for bacterial expression of PTKs, and give an overview on kinase activity assays. Finally, we report our experiences and observations with the kinases Src, Lyn and FAK as examples to illustrate implementation, effects and pitfalls of E. coli expression and in vitro assaying of PTKs.

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“…Wild type human full length TLK1 mammalian expression plasmid was purchased from Addgene (Watertown, MA, USA, cat# 98378). Wild type full length human TLK1B bacterial expression plasmid was obtained from Dr. Sivapriya Kirubakaran, Discipline of Biological Engineering, Indian Institute of Technology, Gandhinagar, India [40]. Dominant negative human TLK1 kinase dead mammalian expression vector was generated as previously described [41].…”

Section: Plasmids and Antibodiesmentioning

confidence: 99%

“…Recombinant full length his-tagged TLK1B and his-tagged MK5 protein was puri ed according to the published literature [12,40].…”

Section: Protein Puri Cationmentioning

confidence: 99%

TLK1-MK5 axis drives prostate cancer cell motility and pathologic features of aggressiveness

Khalil¹,

Singh²,

King³

et al. 2021

Preprint

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Background: Majority of prostate cancer (PCa) related fatalities occur due to metastasis of cancer cells to adjacent and distal organs. We identified the novel interaction between two kinases (TLK1-MK5) that in part may initiate a signaling cascade promoting PCa metastasis. In PCa, TLK1-MK5 signaling might be crucial as androgen deprivation therapy leads to increased expression of TLK1 and compensatory activation of MK5 in metastatic castration-resistant prostate cancer patients. Methods: We performed scratch wound repair and 3D chemotactic migration assays to determine the motility rates of different TLK1 and MK5 perturbed cells. Co-IP, His, and GST pull down, in vitro kinase (IVK) assays and mass spectrometry (MS) were conducted to determine TLK1-MK5 interaction and phosphorylation. Western blotting (WB), immunohistochemistry (IHC) and bioinformatic analysis were used to examine TLK1 and pMK5 levels in PCa cell lines, mice prostate tumors and PCa tissue microarray (TMA). Results: Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 can significantly reduce wound healing rate in MEF and LNCaP cells. However, TLK1 overexpression alone in the MK5 −/− MEF cells did not increase the wound healing which suggested that TLK1 cannot enhance cellular migration in absence of MK5. Our reciprocal co-IPs, His- and GST pull down assays confirmed TLK1-MK5 interaction in cultured cells. Incubation of purified recombinant TLK1B and MK5 increases the phosphorylation of MK5 and its kinase activity. MS analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B. While our WB detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti-androgen treatment increased pMK5 S354 level in a dose-dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells. IHC staining of TRAMP mice prostate tissues also exhibited increased pMK5 S354 level in aggressive tumor compared to benign regions. Finally, IHC analysis of PCa TMA indicated a correlation between elevated pMK5 S354 level and generally higher Gleason scores as well as nodal metastatic status of the tumors. Conclusion: Our data support that TLK1-MK5 signaling is functionally involved in driving PCa cell motility and clinical aggressiveness, hence, disruption of this axis may inhibit the metastasis of PCa.

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High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer

Cited by 16 publications

References 44 publications

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

TLK1-MK5 axis drives prostate cancer cell motility and pathologic features of aggressiveness

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