Phosphorylation of the protein phosphatase 2A inhibitor alpha-endosulfine (ENSA) at two distinct sites, serine 67 and serine 109, in human platelets
E. Walter1, O. Pagel2, R. Zahedi2, A. Smolenski3, S. Gambaryan1,4, U. Walter1, K. Jurk1 (1Mainz, Germany, 2Dortmund, Germany, 3Dublin, Ireland, 4St. Petersburg, Russia)
Platelets - Physiology and Disorders of platelet number and function
Time: 17:15 - 18:15
Objective: The 13 kDa protein alpha-endosulfine (ENSA; or ARPP-19e), originally identified as sulfonylurea receptor ligand, belongs to the highly conserved cAMP-regulated phosphoprotein (ARPP) family with a conserved PKA phosphorylation site (serine 109). In Drosophila and Xenopus, Greatwall kinase phosphorylated ENSA (at serine 67), is a powerful inhibitor of the protein phosphatase 2A (PP2A). In Xenopus, PKA-evoked ENSA S109 phosphorylation keeps the oocyte in prophase whereas S67 phosphorylation is necessary for M-phase entry (1). Very little is known about ENSA in human platelets.
Methods: Proteomic and phosphoproteomic studies with human platelets and platelet proteins were carried out as described (2). ENSA was cloned from human platelets and expressed in HEK293 cells and E.coli BL21. ENSA phosphorylation was studied in washed human platelets and with recombinant proteins.
Results: In our proteomic studies (2) ENSA was detected in human platelets at significant levels (7800 copies/platelet). By our quantitative platelet phosphoproteomic studies, ENSA S109 was found to be strongly phosphorylated (> 10 fold stimulation) in response to cAMP-(Iloprost) and cGMP-elevating (NO-donors, Riociguat) platelet inhibitors (3, unpublished). ENSA S67 phosphorylation was also detected by phosphoproteomics but down-regulated by these platelet inhibitors. Human platelets have two major ENSA transcripts which allowed its cloning and expression in HEK293 cells and E.coli BL21. Wildtype ENSA as well as various ENSA mutants (at S109 and S67) were then generated, purified and tested as PKA or PKG substrates. Further studies with a phosphosite-specific antibody demonstrated an increase of ENSA S67 phosphorylation in platelets in response to the phosphatase inhibitor okadaic acid.
Conclusion: The PP2A phosphatase inhibitor ENSA of human platelets is an excellent PKA and PKG substrate in intact cells and as purified protein. Platelet ENSA S67 phosphorylation was detected for the first time. Ongoing studies aim to identify the ENSA S67 protein kinase and the functional role of this phosphorylation. References 1. Lorca T and Castro A. (2013) Oncogene 32:537-43 2. Burkhart JM et al. (2012) Blood 12: e73-e82 3. Beck F et al. (2014) Blood 123:e1-e10