Identification of a 40 kDa Protein Kinase Activated by Stress in a Halotolerant Green Alga Dunaliella tertiolecta.

Abstract

Activation of a 40 kDa protein kinase in Dunaliella tertiolecta was detected by in-gel kinase assay when the cells were subjected to various forms of stress, such as heat shock, acidic stress and H 2 O 2 . The substrate specificity of the 40 kDa kinase resembles that of HAP kinase (high osmotic pressure-activated protein kinase). An anti-phosphotyrosine monoclonal antibody did not co-precipitate with the 40 kDa kinase in the cell extracts of Dunaliella subjected to stress. Treatment of Dunaliella cells with mastoparan or AlF 4 -, trimeric G-protein activators, stimulated another protein kinase with a relative molecular mass of 46 kDa. This protein kinase has a different substrate specificity from that of HAP kinase and the stress-activated 40 kDa kinase. These results suggest that the stress-activated 40 kDa kinase is closely related to HAP kinase and that the 46 kDa kinase is associated with a putative trimeric G-protein.Key words: Dunaliella, green alga, protein kinase, environmental stress, heat shockThe halotolerant green alga Dunaliella tertiolecta can grow in a medium containing 0.1 to 5 M NaCl. These cells do not have a rigid cell wall and, when subjected to hypo-or hyperosmotic shock, temporarily change shape and volume by regulating the intracellular glycerol content. Recent study implies that enzyme activation via regulatory cofactors or chemical modifications such as phosphorylation/ dephosphorylation may participate in the osmotic stress signaling of Dunaliella 36) .It has been reported that various forms of environmental stress including high osmolarity, heat shock, acidic stress and oxidative stress, induce the activation of corresponding protein kinases, HOG1 in budding yeast cells 21) and MAPKAPkinase-2 20) and SAP kinase/c-Jun kinase 13) in mammalian cells. Recently, evidence has been accumulating that particular protein kinases in plant cells are also activated in response to various environmental stress and phytohormones 10,25) .It was demonstrated that protein phosphorylation is involved in the signal transduction of the halotolerant green alga Dunaliella tertiolecta in response to hypo-and hyperosmotic stress 33,35) . In vivo 32 P-phosphate-labeling experiments using D. tertiolecta cells indicated that hypoosmotic shock causes the transient phosphorylation of the microsomal proteins at 28-33 kDa 36) . A previous study with aequorin, a calcium luminescense protein, and a neutralizing antibody against Dunaliella CDPK (Calcium-Dependent Protein Kinase) indicated that intracellular calcium and CDPK have pivotal roles in the osmoregulation of green algal cells 32) . Hyperosmotic shock rapidly stimulates HAP kinase (High osmotic pressure-Activated Protein kinase) with a relative molecular mass of 40 kDa in Dunaliella cells, while hypoosmotic shock activates LAP kinase (Low osmotic pressure-Activated Protein kinase) of 40 kDa 33) . HAP kinase is regulated by phosphorylation status on its polypeptide and composed of protein kinase cascades involved in the osmosensing system of Dunaliella cells. It has

Two Calcium-Dependent Protein Kinases fromChlamydomonas reinhardtiiare transcriptionally regulated by nutrient starvation

“…47 Almost a decade later, a 40 kDa Ca 2+ -dependent protein kinase was found to be activated in Dunaliella tertiolecta by heat shock, acidic stress, and H 2 O 2 treatment. 48 Using an in-gel kinase assay, 3 CDPKs (62, 54, and 47 kDa) were identified in D. tertiolecta cell extracts. 40 When the same technique was extended to homogenates of another green alga, Closterium ehrenbergii using histone H1, myelin basic protein, and casein as substrates, CDPKs ranging in relative molecular mass from 47-60 kDa were observed.…”

A Calcium-dependent Protein Kinase (CDPK) in the Unicellular Green Alga Closterium ehrenbergii

“…The protein kinase activity in algal samples was detected by the in-gel protein kinase method described by Kameshita and Fujisawa 13) with minor modifications 14) . Briefly, the protein substrate-0.2 mg/mL of histone type III-S (histone H1), myelin basic protein (MBP), or casein-was immobilized on 10% polyacrylamide gels polymerized as for conventional SDS-PAGE 15) .…”