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Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle
Abstract:
Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.
Insights
Cyclic AMP stimulates the phosphorylation of filamin, an actin-binding protein, in guinea pig vas deferens smooth muscle. This contrasts with other proteins whose phosphorylation is regulated by cyclic GMP.
Area of Science:
- Biochemistry
- Cell Biology
- Muscle Physiology
Background:
- Filamin is a high molecular weight actin-binding protein abundant in smooth and non-muscle cells.
- Understanding protein phosphorylation in smooth muscle is crucial for elucidating contractile mechanisms.
Purpose of the Study:
- To investigate the phosphorylation of filamin in mammalian smooth muscle (guinea pig vas deferens).
- To compare the effects of cyclic AMP (cAMP) and cyclic GMP (cGMP) on protein phosphorylation in smooth muscle.
Main Methods:
- In vivo phosphorylation studies using [32P]orthophosphate in intact vas deferens.
- In vitro phosphorylation assays with [gamma-32P]ATP on particulate fractions.
- Characterization of kinase activity and substrate specificity using purified proteins.
Main Results:
- Filamin phosphorylation in vas deferens was stimulated by cAMP, but not by cGMP or Ca2+.
- Purified filamin was phosphorylated by cAMP-dependent protein kinase.
- cGMP stimulated the phosphorylation of two particulate proteins (G-I and G-III), unlike filamin.
Conclusions:
- Filamin phosphorylation in smooth muscle is regulated by a cAMP-dependent pathway.
- Distinct signaling pathways involving cAMP and cGMP regulate different protein phosphorylation events in smooth muscle.