Functional Basis and Biophysical Approaches to Characterize the C-Terminal Domain of Human-Ribosomal S6 Kinases-3.
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.
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, Kharghar, 410 210, Maharashtra, India.
Bhanu P
BP
Jagilinki
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, Kharghar, 410 210, Maharashtra, India.
Rajan Kumar
RK
Choudhary
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, Kharghar, 410 210, Maharashtra, India.
Pankaj S
PS
Thapa
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, Kharghar, 410 210, Maharashtra, India.
Nikhil
N
Gadewal
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, Kharghar, 410 210, Maharashtra, India.
M V
MV
Hosur
Department of Biochemistry and Bioinformatics Centre, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, 442102, India.
Satish
S
Kumar
Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai, Kharghar, 410 210, Maharashtra, India. avarma@actrec.gov.in.
Ashok K
AK
Varma
D002942:Circular Dichroism; D003001:Cloning, Molecular; D000067493:Dynamic Light Scattering; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D062105:Molecular Docking Simulation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D048049:Extracellular Signal-Regulated MAP Kinases
CD spectroscopy; Dynamic light scattering; Fluorescence spectroscopy; In silico modelling; Inclusion bodies; MAPK/ERK and RSK