Identification of cGMP-dependent protein kinase and its specific substrates in the anterior pituitary.

摘要

In the anterior pituitary, cGMP is produced in response to a number of stimuli, but intracellular events distal to cGMP production are obscure. Since cGMP-dependent protein kinase (PKG) is a major effector of cGMP actions in other tissues we have determined whether PKG and its specific substrates might be present and responsive to external signals in the ovine anterior pituitary. Photoaffinity labelling with [32P]cGMP revealed a specific 78 kDa protein in ovine anterior pituitary that comigrated with purified bovine lung PKG-I. PKG in protein extracts from anterior pituitary or cultured anterior pituitary cells was enriched by DEAE ion-exchange chromatography and assayed for activity. Both tissue and cultured cells had a relatively high PKG activity by comparison with aortic smooth muscle (known high activity) and brain (known low activity). Subcellular distribution studies showed that in anterior pituitary, aortic and brain, PKG activity was present in both cytosol and triton-extracted membrane fractions, while in platelets the activity was associated with only the membrane fraction. To determine if this PKG might be responsive to extracellular signals an activity ratio assay was used. Incubation of cultured cells with atrial natriuretic peptide (ANP) and sodium nitroprusside, activators of membrane and cytosolic guanylate cyclases respectively, increased the activity of PKG. To determine events distal to PKG activation, a search for potential substrates of PKG was performed. Few substrates were detectable upon addition of purified PKG to tissue lysates due to the high background activity of endogenous protein kinases in the anterior pituitary. However, 19 substrates of PKG were detected in heat-stable and 14 in acid-soluble protein extracts of the anterior pituitary, in which background phosphorylation was almost abolished. After partial purification through Q-Sepharose ion-exchange chromatography some of these proteins were preferentially phosphorylated by addition of PKG-I, while the others were additionally substrates of exogenous cAMP-dependent protein kinase (PKA) or Ca2+ and phospholipid-dependent protein kinase (PKC). A 132-kDa substrate showed an identical phosphopeptide map to a PKG substrate previously described in vascular smooth muscle and platelets. These data demonstrate for the first time the presence of functional PKG activity and multiple PKG substrates in the anterior pituitary where they may play a role in mediating the intracellular actions of cGMP.