Abstract
Protein kinase G type II (PKG II) is a serine/threonine-protein kinase that was originally isolated from the small intestinal mucosa with primary functions in the secretion of small intestinal mucosal cells, secretion of renin and aldosterone, and chondrocyte activities. Recent studies have shown that PKG II exerts anti-tumor effects, while a previous study by our group confirmed that PKG II inhibited the proliferation and migration of cancer cells. Interestingly, PKG II, which was typically bound to the intracellular side of the membrane, was detected in the serum and cell culture medium as a diagnostic biomarker of tumor growth. Thus, the aim of the present study was to elucidate the function and the targets of PKG II, and the mechanism underlying the secretion of this kinase. Construction of peptides and plasmids, RNA interference, Immunoelectron microscopy, Co-immunoprecipitation, N-glycosylation assay and Isolation of the Golgi apparatus were applied to investigate the secretory mechanism, and the targets and function of PKG II. PKG II was secreted by enterochromaffin (EC) cells, which were components of the endocrine system in the gastrointestinal tract. Myristoylation of glycine 2 and the N-terminal sequence, especially the amino acids 3-30, acted as a signal peptide to induce the secretion of PKG II via the conventional protein secretory pathway. Moreover, recombinant PKG II inhibited the epidermal growth factor (EGF)-induced activation of the EGF receptor via phosphorylating the T406 of the extracellular domain and blocked EGF-triggered proliferation of various cancer cells. These results revealed a correlation between the endocrine system and the secretion of protein kinase, suggesting a novel protein secretory pathway. The resuls also indicated that secreted PKG II was a potential diagnostic biomarker and an inhibitor of tumor.
Highlights
Protein Kinases are a heterogeneous family of enzymes modulating several biological pathways, including cell survival, differentiation and apoptosis [1]
The confocal microscopy results showed that there is colocalization of PKG Protein kinase G type II (II) and Chromogranin A (CGA) in the same cell (Fig. 2A), hinting that Protein kinase G type II (PKG II) might be secreted by EC cells and convey endocrinological activities
The results of immunoelectron microscopy confirmed that cells in GI tissues had secretory vesicles that contained CGA, while other secretory vesicles contained PKG II (Fig. 2B)
Summary
Protein Kinases are a heterogeneous family of enzymes modulating several biological pathways, including cell survival, differentiation and apoptosis [1]. Our group confirmed that PKG II could inhibit the proliferation and migration of gastric cancer cells by blocking epidermal growth factor (EGF)-induced activation of the epidermal growth factor receptor (EGFR) [10,11]. The aim of the present study was to elucidate the function and the targets of PKG II, and the mechanism underlying the secretion of this kinase. Myristoylation of glycine 2 and the N-terminal sequence, especially the amino acids 3–30, acted as a signal peptide to induce the secretion of PKG II via the conventional protein secretory pathway. Recombinant PKG II inhibited the epidermal growth factor (EGF)induced activation of the EGF receptor via phosphorylating the T406 of the extracellular domain and blocked EGF-triggered proliferation of various cancer cells. The resuls indicated that secreted PKG II was a potential diagnostic biomarker and an inhibitor of tumor
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