Abstract
We previously reported that when 32Pi-loaded rat parotid slices are incubated with the beta-adrenergic agonist isoproterenol, the level of a soluble 32P-labeled 17-kDa protein (pp17) decreases rapidly (Kanamori, T., and Hayakawa, T. (1982) Biochem. Int. 4, 517-523). Here we show that pp17 consists of two distinct phosphoproteins (pp17a and pp17b), identify their unphosphorylated forms (p17a and p17b, respectively), and provide evidence for their beta-adrenergic stimulation-induced dephosphorylation. Since p17a and p17b were predominant forms even in nonstimulated cells, peptides were generated from them with Staphylococcus aureus V8 protease or cyanogen bromide; subsequent sequencing of these peptides and homology search allowed identification of p17a and p17b as destrin- and cofilin-like proteins, respectively. Interestingly, they were also dephosphorylated in response to cholinergic stimulation. Because destrin and cofilin are actin-depolymerizing proteins whose activities are possibly regulated by their phosphorylation/dephosphorylation, the two parotid proteins reported here might be involved in cortical F-actin disruption observed in parallel with exocytotic amylase secretion.
Highlights
We previously reported that when 32Pi-Ioaded rat parotid slices are incubated with the /.:I-adrenergic agonist isoproterenol, the level of a soluble 32P-Iabeled 17-kDa protein decreases rapidly (Kanamori, T., and Hayakawa, T. (1982) Biochem
Because destrin and cofilin are actin-depolymerizing proteins whose activities are possibly regulated by their phosphorylation/dephosphorylation, the two parotid proteins reported here might be involved in cortical F-actin disruption observed in parallel with exocytotic amylase secretion
In accordance with this view, there exist several lines of evidence suggesting the involvement of cAMP-dependent protein kinase in l3-adrenergic agonist-induced amylase secretion, and endogenous protein substrates for this enzyme have extensively been studied with 32P;-loaded rat parotid slices or cell aggregates
Summary
Vol 270, No 14, Issue of April 7, pp. 8061-8067, 1995 Printed in U.S.A. Identification of Two 17-kDa Rat Parotid Gland Phosphoproteins, Subjects for Dephosphorylation upon Jl-Adrenergic Stimulation, as Destrin- and Cofilin-like Proteins*. Since pl7a and pl7b were predominant forms even in nonstimulated cells, peptides were generated from them with Staphylococcus aureus V8 protease or cyanogen bromide; subsequent sequencing of these peptides and homology search allowed identification of pl7a and pl7b as destrin- and cofilin-like proteins, respectively. They were dephosphorylated in response to cholinergic stimulation. We show that our 17-kDa soluble phosphoprotein (ppI7) consists of two distinct phosphoproteins, identify them as phosphorylated forms of destrin- and cofilin-like proteins, provide evidence for their l3-adrenergic stimulation-induced dephosphorylation, and discuss their possible involvement in parotid amylase secretion
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