Study On The Secretion Mechanism Of Type II CGMP-dependent Protein Kinase

Protein secretion is an important physiological activity.The secretory pathways of proteins are divided into classical secretory pathway(endoplasmic reticulum-Golgi pathway,ER-Golgi pathway)and nonclassical pathways.At present,the research achievements of protein secretion are not significant,and reported secretory proteins are very limited,whose secretion pathways and molecular mechanisms are not clear.With the deepening of research,some secreted proteins discovered do not belong to established classical or nonclassical secretory proteins,and these findings will be important for the study of protein secretion.Type Ⅱ cGMP-dependent protein kinase(PKG Ⅱ)is a serine / threonine protein kinase whose expression and activity in tumor tissue and tumor cell lines is very low and found that it is associated with the occurrence and development of tumor.It was found that PKG Ⅱ existed in the culture supernatants of gastric cancer cell line HGC-27 and cervical cancer cell line Hela infected with the adenovirus Ad-PKG Ⅱ.Then it was found that PKG Ⅱ also existed in human and mouse serum by ELISA.These results indicated that PKG Ⅱ was a secreted protein.In the following,this subject delved into the secretion pathway of PKG Ⅱ.The amino acid sequence of PKG Ⅱ was analyzed by software SignalP 4.1 Server.The results showed that PKG Ⅱ had no classical signal peptide sequence,suggesting that PKG Ⅱ should belong to nonclassical secretory protein.The co-localization of PKG Ⅱ and lysosome-associated membrane protein 1(LAMP1),lysosomal marker,was not observed in HGC-27 cells by laser confocal microscopy,and PKG Ⅱ did not exist in the exosomes.PKG Ⅱ did not secrete through the two kinds of nonclassical pathways----lysosomal pathway and exosomes pathway.Based on PKG Ⅱ located on the side of the nucleus,similar to the distribution of ER and Golgi in cells,the co-localization of PKG Ⅱ and ER marker protein Glucose-6-phosphatase(G-6-Pase)/ Golgi marker protein 130 kDa cis Golgi matrix protein(GM130)in both the cell and the human small intestine were observed by laser confocal microscopy,and it was found that PKG Ⅱ was co-located with the ER / Golgi in both.To further demonstrate that PKG Ⅱ was secreted through the ER-Golgi pathway,the following experiments were conducted: in vitro and in vivo experiments were performed with Brefeldin A(BFA),an inhibitor of the ER-Golgi pathway,and the interaction between PKG Ⅱ and ER protein 78 kDa glucose-regulated protein(GRP78)was detected by co-immunoprecipitation(Co-IP).The results showed that BFA could block the secretion of PKG Ⅱ in cells and mice,and PKG Ⅱ was bound with GRP78.PNGase F enzyme hydrolysis was used to detect the N-glycosylation of PKG Ⅱ,and the result showed that the molecular weight of PKG Ⅱ in the digested group decreased,indicating that the intracellular PKG Ⅱ underwent N-glycosylation modification.The Golgi were extracted from the cells and it was found that PKG Ⅱ was present in the Golgi apparatus.These results demonstrated that PKG Ⅱ was secreted by classical pathway.Then this research focused on the mechanism of PKG Ⅱ entry into the ER.In view of PKG Ⅱ being myristoylated protein,the 2-glycine in PKG Ⅱ N-terminal was mutated to alanine which could not be myristoylated,and it was found that 2-glycine mutation could change the distribution of PKG Ⅱ in the cells.The adenovirus Ad-PKG Ⅱ-G2 A was constructed and the content of PKG Ⅱ in the culture supernatant was detected and the co-localization of PKG Ⅱ-G2 A and G-6-Pase / GM130 were observed.The results showed that the cells did not secrete PKG Ⅱ-G2 A,and it was not co-located with the ER/ Golgi,i.e,it was no longer through this secretion pathway.The above studies showed that the myristoylation of PKG Ⅱ could affect its secretion,and the myristoyl modification of the protein was caused by myristoyltransferase.Next,the relationship between the myristoyl CoA: protein N-myristoyl transferse 1(NMT-1)and PKG Ⅱ was studied.It was detected that PKG Ⅱ combined with NMT-1,and PKG Ⅱ and NMT-1 were co-localization in HGC-27 cells and human gastrointestinal specimens.To further demonstrate that the myristoylation of PKG Ⅱ could affect its secretion,and then in vitro and in vivo experiments were conducted with myristoyltransferase inhibitor Tris(dibenzylideneacetone)-dipalladium(DBA)and 2-Hydroxymyristic Acid(HMA).In vitro and in vitro experiment: ELISA results showed that PKG Ⅱ in mice serum and the supernatant decreased significantly;i.e,the myristoylation of PKG Ⅱ could affect the secretion of PKG Ⅱ in mice and in cells.Based on the results and the literature,it was speculated that the myristylation of the 2-glycine of PKG Ⅱ could affect hydrophobicity of the peptide,thereby affecting its binding to the ER membrane and its entry into the ER-Golgi secretion pathway.Next,the amino acid sequence affecting the secretion of PKG Ⅱ would be clarified.Signal recognition particle 54(SRP54)recognition with signal peptide is a key step in guiding neonatal peptides to ER.In this study,the expression of SRP54 in cells interfered with siRNA was observed.The results showed that PKG Ⅱ in cell supernatant decreased with the increase of interference,which indicated that the secretion of PKG Ⅱ was affected by SRP54.PKG Ⅱ combined with SRP54,suggesting that PKG Ⅱ did exist a sequence for SRP54 identification and combination.On the basis of retention of 2-glycine,the N-terminal of PKG Ⅱ was subjected to sequence knockout experiments,and seven deletants were constructed.The results showed that the cells transfected with plasmid PKG Ⅱ-Del3-30-flag did not secrete PKG Ⅱ and it was not co-located with GM130.It was found that the binding ability of PKG Ⅱ to SRP54 was largly reduced by Co-IP.The above results indicated that PKG Ⅱ was recognized and bound by SRP54 through the 3-30 amino acids sequence,which led to its entry into the ER-Golgi pathway.Finally,the sequence of signal peptide action in PKG Ⅱ should be clarified.Combined with the 2-glycine myristoylation modification and the 3-30 amino acids sequence of PKG Ⅱ,the binding ability of PKG Ⅱ-flag,PKG Ⅱ-Del3-30-flag and PKG Ⅱ-G2A-flag to SRP54 was detected by immunoprecipitation.The results showed that the binding of PKG Ⅱ and SRP54 was significantly attenuated and similar in the cells transfected with 3-30 amino acids deletant or 2-glycine mutant.This indicated that 2-30 amino acids of PKG Ⅱ might act as signal sequence.In view of the role of starting amino acid,plasmid and peptides of PKG Ⅱ 1-30 amino acids were constructed to verify,respectively.Immunoprecipitation results showed that SRP54 could be detected in IP group,indicating that PKG Ⅱ-N30 AA could bind SRP54 and the content of SRP54 in IP group: PKG Ⅱ-N30AA-flag polypeptide group >PKG Ⅱ-N30AA-flag polypeptide group >PKG Ⅱ-N30AA-flag polypeptide group + HMA,which further showed that the myristoylated PKG Ⅱ-N30 AA favored its combination with SRP54,that was more conducive to its secretion.Thus it was determined that the 2-glycine myristoylation of PKG Ⅱ and the role of the 3-30 amino acids sequence was co-acting as a signal peptide.Next it was explored whether this amino acid sequence could lead to the secretion of heterologous protein.The results showed that the distribution of GFP in the transfected PKG Ⅱ-N30AA-GFP plasmid group changed,and co-localization with GM130 occurred.This also demonstrated that PKG Ⅱ-N30 AA could lead GFP into ER-Golgi secretion as signal peptide sequence.Some of the abundant proteins expressed in the cells from hundreds of myristoylated proteins were selected to detect their secretion.It was found that flap endonuclease 1(FEN1)and cAMP were dependent(cAMP-dependent protein kinase,PKA)belonged to the secretory myristoylated protein,while the myristoylated alanine-rich protein kinase C substrate and the serine /(3’-kinase regulatory subunit 4,PIK3R4 / Vps15)and 2’-5’-oligoadenylate synthase 2(2’-5’-oligoadenylate synthetase 2,OAS2))were non-secreted myristoylated proteins,and the nitric oxide synthase(NOS3)was a selective secretory protein.The results indicated that the mechanism of protein secretion was very complex,and myristoylation might only be one of the conditions of myristoylated protein secretion,and the secretion should also be related to its amino acids sequence.The expressions of FEN1 and PKA in the supernatants of the cells significantly decreased with the increase of SRP54-siRNA interference.The results showed that the secretion of FEN1 and PKA was affected by SRP54.It was suggested that FEN1 and PKA could be studied in detail to follow the mechanism of PKG Ⅱ secretion,which would be helpful to the study of myristoyl protein secretion.In summary,PKG Ⅱ is a secreted protein,and its 2-glycine myristoylated modification and the 3-30 AA sequence can act as a signal peptide,and then guide its access to ER-Golgi secretion pathway.