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

Sujata Chauhan; Subhasis Samanta; Nitin Sharma; Jitendra K Thakur; Kamal Dev; Anuradha Sourirajan

doi:10.1016/j.heliyon.2019.e03050

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

Heliyon. 2019 Dec 24;5(12):e03050. doi: 10.1016/j.heliyon.2019.e03050. eCollection 2019 Dec.

Authors

Sujata Chauhan¹, Subhasis Samanta², Nitin Sharma¹, Jitendra K Thakur², Kamal Dev¹, Anuradha Sourirajan¹

Affiliations

¹ Faculty of Applied Sciences and Biotechnology, Shoolini University of Biotechnology and Management Sciences, Bajhol, PO Sultanpur, District Solan, Himachal Pradesh, 173229, India.
² National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi, 110067, India.

Abstract

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 K_M 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.

Keywords: Biochemistry; Biological sciences; Cancer research; Cdc5; Cell biology; GST-Cdc5; Health sciences; In vitro kinase assay; Mg2+; Oncology; PLK; Proteins; Saccharomyces cerevisiae.