Purification, identification, and biochemical characterization of a fatty-acid-stimulated Ser/Thr protein phosphatase
Abstract
An arachidonic acid-stimulated protein Ser/Thr phosphatase activity was detected in soluble extracts prepared from clonal GH4C 1 rat pituitary cells, rat or bovine brain, and bovine heart. The lipid-stimulated Ser/Thr phosphatase was isolated from soluble extracts of rat or bovine brain as an active monomer. The purified bovine enzyme migrates as a single 63 kDa polypeptide, and was identified by sequence analysis to be a closely related homolog of the recently cloned protein Ser/Thr phosphatase 5 (PP5). Protein phosphatase 5 and its yeast homolog, PPT1, contain a C-terminal catalytic domain that is structurally related to the catalytic subunits of PP2A and PP1, and an N-terminal domain consisting of several tetratricopeptide repeats that are unique among known protein Ser/Thr phosphatases. Although mammalian PP5 was previously cloned, little is known about its enzyme activity or potential for regulation. Arachidonic acid stimulates the purified bovine PP5 activity towards a variety of phosphosubstrates in vitro, suggesting that the lipid effect is enzyme-directed. Lipid stimulation ranged from 2–14-fold depending on the concentration of lipid, with half-maximal stimulation occurring at approximately 35–100 [special characters omitted]M AA. Similar activation is observed with other unsaturated fatty acids, but not by alcohol or methyl ester analogs or saturated fatty acids, suggesting that a polar head group and at least one double bond are required for the lipid effect. Recombinant rat PP5 exhibits a specific activity and sensitivity to arachidonic acid similar to that of the purified bovine brain enzyme. At an ionic strength similar to physiologic conditions, recombinant PP5 was stimulated 40-fold by AA. The stimulation of the recombinant enzyme by AA is consistent with our identification of the lipid-stimulated phosphatase from bovine brain as a homolog of PP5. In addition, we find that recombinant and native PP5 are active and stimulated by AA in the presence of Mn2+, Ni 2+, or Mg2+ toward para-nitrophenylphosphate and phosphotyrosine substrates. In summary, the activation of PP5 by unsaturated fatty acids reveals the potential of PP5 regulation, and raises the possibility of its control in vivo by lipid second messengers or another endogenous activator.
Degree
Ph.D.
Advisors
Rossie, Purdue University.
Subject Area
Biochemistry|Molecular biology
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