Somlata, * and Kamanna, Sathisha and Agrahari, Mridula and Babuta, Mrigya and Bhattacharya, Sudha and Bhattacharya, Alok (2012) Autophosphorylation of Ser(428) of EhC2PK Plays a Critical Role in Regulating Erythrophagocytosis in the Parasite Entamoeba histolytica. In: Journal of Biological Chemistry, 287 (14). pp. 10844-10852.
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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 Mn2+-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, KD Delta C) 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 KD Delta C 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.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article is belongs to The American Society for Biochemistry and Molecular Biology.|
|Keywords:||ACTIVATION-LOOP AUTOPHOSPHORYLATION; PROTEIN-KINASE-C; PHAGOCYTOSIS;BINDING;VIRULENCE;DYNAMICS;DOMAIN;CELLS|
|Department/Centre:||Division of Biological Sciences > Molecular Biophysics Unit|
|Date Deposited:||11 Aug 2012 07:26|
|Last Modified:||11 Aug 2012 07:26|
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