title: Autophosphorylation of Ser428 of EhC2PK plays a critical role in regulating erythrophagocytosis in the parasite Entamoeba histolytica.
abstract: The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn(2+)-dependent serine kinase. It has a trans-autophosphorylated site at Ser(428) as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KDΔC) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KDΔC proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica.
authors: School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
authors: Somlata
authors: Sathisha
authors: S
authors: Kamanna
authors: Mridula
authors: M
authors: Agrahari
authors: Mrigya
authors: M
authors: Babuta
authors: Sudha
authors: S
authors: Bhattacharya
authors: Alok
authors: A
authors: Bhattacharya
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019295:Computational Biology; D004748:Entamoeba histolytica / Q000166:cytology* / Q000201:enzymology*; D004912:Erythrocytes / Q000469:parasitology*; D008969:Molecular Sequence Data; D010587:Phagocytosis; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D012694:Serine; D000255:Adenosine Triphosphate; D011494:Protein Kinases