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Functional Basis and Biophysical Approaches to Characterize the C-Terminal Domain of Human—Ribosomal S6 Kinases-3

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Abstract

Ribosomal S6 kinases (RSKs) are the major functional components in mitogen-activated protein kinase (MAPK) pathway, and these are activated by upstream Extracellular signal-regulated kinase. Upon activation, RSKs activate a number of substrate molecules involved in transcription, translation and cell-cycle regulation. But how cellular binding partners are engaged in the MAPK pathways and regulate the molecular mechanisms have not been explored. Considering the importance of protein–protein interactions in cell signalling and folding pattern of native protein, functional C-terminal kinase domain of RSK3 has been characterized using in vitro, in silico and biophysical approaches. RSKs discharge different functions by binding to downstream kinase partners. Hence, depending upon cellular binding partners, RSKs translocate between cytoplasm and nucleus. In our study, it has been observed that the refolded C-terminal Kinase domain (CTKD) of RSK 3 has a compact domain structure which is predominantly α-helical in nature by burying the tryptophans deep into the core, which was confirmed by CD, Fluorescence spectroscopy and limited proteolysis assay. Our study also revealed that RSK 3 CTKD was found to be a homotrimer from DLS experiments. A model was also built for RSK 3 CTKD and was further validated using PROCHECK and ProSA webservers.

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Abbreviations

RSK:

Ribosomal s6 kinase

MAPK:

Mitogen-Activated Protein Kinase

ERK:

Extracellular signal-regulated kinase

PPI:

Protein–Protein Interactions

CTKD:

C-terminal kinase Domain

IPTG:

Isopropyl-β-D-thiogalactoside

DLS:

Dynamic Light Scattering

IMAC:

Immobilized Metal ion Affinity Chromatography

RMSD:

Root Mean Square Deviation

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Acknowledgments

We thank CRI common facility, DBT funded BTIS, Proteomics facility at ACTREC. Mr Bhanu thanks UGC, New Delhi for fellowship. Dr. M.V. Hosur is thankful to DAE for RRF award.

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Authors and Affiliations

  1. Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, Kharghar, 410 210, Maharashtra, India

    Bhanu P. Jagilinki, Rajan Kumar Choudhary, Pankaj S. Thapa, Nikhil Gadewal, M. V. Hosur & Ashok K. Varma

  2. Department of Biochemistry and Bioinformatics Centre, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, 442102, India

    Satish Kumar

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  1. Bhanu P. Jagilinki
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  2. Rajan Kumar Choudhary
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Correspondence to Ashok K. Varma.

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Jagilinki, B.P., Choudhary, R.K., Thapa, P.S. et al. Functional Basis and Biophysical Approaches to Characterize the C-Terminal Domain of Human—Ribosomal S6 Kinases-3. Cell Biochem Biophys 74, 317–325 (2016). https://doi.org/10.1007/s12013-016-0745-6

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  • DOI: https://doi.org/10.1007/s12013-016-0745-6

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