Record Details

Saccharomyces cerevisiae polo-like kinase, Cdc5 exhibits ATP-dependent Mg2+ -enhanced kinase activity in vitro

NIPGR Digital Knowledge Repository (NDKR)

View Archive Info
 
 
Field Value
 
Title Saccharomyces cerevisiae polo-like kinase, Cdc5 exhibits ATP-dependent Mg2+ -enhanced kinase activity in vitro
 
Creator Chauhan, Sujata
Samanta, Subhasis
Sharma, Nitin
Thakur, Jitendra K.
Dev, Kamal
Sourirajan, Anuradha
 
Subject Biological sciences
Cell biology
Proteins
Biochemistry
Cancer research
Health sciences
Oncology
PLK
Cdc5
Saccharomyces cerevisiae
GST-Cdc5
In vitro kinase assay
Mg2+
 
Description Accepted date: 11 December 2019
Phosphorylation of proteins on serine/threonine residues represents an important biochemical mechanism to regulate several cellular processes. Polo-like kinases (PLKs) are a family of serine-threonine kinases that play an imminent role in cell cycle regulation in yeast to humans, and thus an important therapeutic target for cancers. The present study provides insights into the enzymatic features of Saccharomyces cerevisiae PLK, Cdc5 using in vitro casein phosphorylation assays. The recombinant yeast PLK, GST-Cdc5 showed maximum casein phosphorylation activity at 30 °C, pH 9 and 45 min of incubation period. GST-Cdc5 exhibited a KM of 1.35 μM for casein, and high affinity for ATP, since addition of non-radioactive ATP chased out casein phosphorylation by radiolabeled ATP. The recombinant enzyme showed maximum kinase activity at 2.7 μM of GST-Cdc5. Casein was found to be the best in vitro substrate of GST-Cdc5 followed by BSA (Bovine Serum Albumin) and MBP (Myelin Basic Protein). Of the metal ions tested, Mg2+ (at 20 mM) was found to enhance GST-Cdc5 kinase activity, while Ca2+ (at 5 mM) and Mn2+ (at 10 mM) inhibited the same. The presence of EDTA, SDS and PMSF inhibited phosphorylation by GST-Cdc5, while DTT had no effect. The recombinant GST-Cdc5 can be used as a tool for deciphering PLKs’ structure and functions, which are still at infancy.
The authors would like to acknowledge Shoolini University, Solan, for
the infrastructural support and NIPGR, New Delhi for the assistance
provided in this study. The authors also thank Divyanshi Sharma for
assistance in hydropathy analysis.
 
Date 2019-12-31T06:08:58Z
2019-12-31T06:08:58Z
2019
 
Type Article
 
Identifier Heliyon 5(12): e03050
2405-8440
http://223.31.159.10:8080/jspui/handle/123456789/1027
https://www.sciencedirect.com/science/article/pii/S240584401936709X
 
Language en_US
 
Format application/pdf
 
Publisher Elsevier B.V.