PKG-Mediated Protein Phosphorylation

If one role of PKG is to decrease smooth muscle intracellular Ca levels, then the mechanism by which this occurs is still not completely defined. Several protein substrates that are known to be phosphorylated by activation of PKG have been identified in SMCs, platelets, and other tissues. Interestingly, some of these proteins have been shown by others to regulate intracellular Ca levels in various cell types. For example, phospholamban, when phosphorylated by PKA in cardiac myocytes, dissociates from sarcoplasmic reticulum (SR) Ca -ATPase resulting in the enhanced sequestration of Ca into the SR (Lindemann et al., 1983). A similar effect of PKG activation has been shown in SMCs (Cornwell et al., 1991 Karczewski et al., 1992 Raeymaekers et al., 1988 Sarcevic et al., 1989). Using another example, Supattopone et al. (1988) reported that PKA-dependent phosphorylation of the inositol 1,4,5-trisphosphate (IP3) receptor in broken-cell fractions decreased Ca release from brain microsomes. Our laboratory has recently shown that the IP3 receptor is also phosphorylated by PKG in vitro on the major PKA phosphorylation site (i.e., Ser-1755) (Komalavilas and Lincoln, 

To determine whether the IP3 receptor protein is phosphorylated in the intact cell by PKG, we used the technique of back-phosphorylation  

FIGURE 3 Effects of 8-/ -chlorophenylthio-cAMP and -cGMP (8-CPT-cAMP and 8-CPT-cGMP, respectively) on the phosphorylation of the inositol 1,4,5-trisphosphate (IP3) receptor in rat aorta. Rat aortic strips were incubated for 10 min with 50 ftM of the cyclic nucleotide analogs, then the IP3 receptor was solubilized and immunoprecipitated, and phos-phorylated in vitro using purified PKG and [y- P]ATP. Following separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, endogenous phosphorylation of the IP3 receptor was estimated by back-phosphorylation.  

Although the role of the phosphorylation of the IP3 receptor is not known at this time, these results indicate that phosphorylation of proteins by cyclic nucleotide-dependent protein kinases is a more complex event than the mere recognition of substrates by kinases via phosphorylation sites on the substrate. Our laboratory has suggested that the localization of PKG with substrates (which may or may not be specific substrate proteins for PKG in vitro) may contribute to the selective phosphorylation of proteins by this kinase (Cornwell et al., 1991 Lincoln, 1994 Wyatt et al 1991). Hence, the proteins localized to the rat aortic SMC SR (phospholamban and the IP3 receptor) are selectively phosphorylated upon elevation of cGMP, but not cAMP, due to the localization of PKG to this region of the cell. It is the localization of the protein kinase to substrate that may determine the physiological outcome of phosphorylation in the intact cell. This point is illustrated in Fig. 4. In Fig. 4B PKG is proposed to be localized with specific proteins in the rat aortic SMC SR (i.e., phospholamban and the IP3 receptor). PKA is not found in this organelle (Cornwell et al., 1991). Elevations in 

FIGURE 5 Immunoprecipitation of vimentin by anti-protein kinase G (PKG) from rat aortic smooth muscle cells (SMCs). PKG was immunoprecipitated from low-passaged (passage 1) SMCs the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Vimentin was visualized using mouse antivimentin in a Western blot. The antibody also recognizes isotopes of rabbit anti-bovine PKG, as indicated by the staining of the immunoglobulin G heavy chain. [Data from MacMillan-Crow and Lincoln (1994) with permission of the American Chemical Society.] 

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