title: Activation of the 43 kDa inositol polyphosphate 5-phosphatase by 14-3-3zeta.
abstract: The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyzes and thereby inactivates the second messenger molecules inositol 1,4,5-trisphosphate -Ins(1,4,5)P3- and inositol 1,3,4,5-tetrakisphosphate in a signal terminating reaction. Recent studies have shown that the platelet protein pleckstrin forms a complex with the 43 kDa 5-phosphatase and activates Ins(1,4,5)P3 hydrolysis 2-fold [Auethavekiat, V., Abrams, C. S., & Majerus, P. W. (1997) J. Biol. Chem. 272, 1786-1790]. We now show that another platelet protein, 14-3-3zeta, forms a complex with the 43 kDa 5-phosphatase and thereby activates the hydrolysis of Ins(1,4,5)P3. Both pleckstrin and 14-3-3zeta contain one or more pleckstrin-homology domains, both are present in platelet cytosol, and both dimerize and form complexes with other signalling proteins. Purified platelet pleckstrin and 14-3-3zeta enhanced the rate of the hydrolysis of Ins(1,4,5)P3 by the 43 kDa 5-phosphatase 1.9- and 3.8-fold, respectively, but did not activate the 75 kDa 5-phosphatase. We have demonstrated that the mechanism of 5-phosphatase activation by 14-3-3zeta results from specific complex formation between the 43 kDa 5-phosphatase and 14-3-3zeta. Recombinant 43 kDa 5-phosphatase bound to recombinant glutathione S-transferase (GST)/14-3-3zeta fusion protein, but not GST alone, immobilized on glutathione-Sepharose. A potential 14-3-3 binding motif was located in the 43 kDa, but not the 75 kDa, 5-phosphatase. The motif "363RSESEE" is present in close proximity to the proposed catalytic domain of the 43 kDa 5-phosphatase. A synthetic peptide corresponding to the putative 14-3-3 binding motif demonstrated specific, saturable binding to purified 125I-14-3-3, with a Kd of 92 nM. In addition, platelet cytosolic 5-phosphatase bound to recombinant 14-3-3zeta immobilized on glutathione-Sepharose. Thus, 14-3-3zeta serves in human platelets to activate the 43 kDa 5-phosphatase and may thereby function to prevent generation of Ins(1,4,5)P3 -mediated calcium release in unstimulated platelets.
authors.affiliation: Monash University, Department of Medicine, Box Hill Hospital, Nelson Road, Box Hill, Melbourne, Victoria, Australia 3128.
authors.forename: J K
authors.initials: JK
authors.lastname: Campbell
authors.forename: R
authors.initials: R
authors.lastname: Gurung
authors.forename: S
authors.initials: S
authors.lastname: Romero
authors.forename: C J
authors.initials: CJ
authors.lastname: Speed
authors.forename: R K
authors.initials: RK
authors.lastname: Andrews
authors.forename: M C
authors.initials: MC
authors.lastname: Berndt
authors.forename: C A
authors.initials: CA
authors.lastname: Mitchell
mesh_terms: D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D000072181:Inositol Polyphosphate 5-Phosphatases; D007700:Kinetics; D010750:Phosphoproteins; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
chemical_list: D048948:14-3-3 Proteins; D001798:Blood Proteins; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D014446:Tyrosine 3-Monooxygenase; D005982:Glutathione Transferase; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases