Proteomic identification of posttranslational modifications: cAMP-induced changes of phosphorylation and investigation of novel approaches detecting posttranslational modifications at lysine and arginine residues

Abstract

Cyclic adenosine monophosphate (cAMP) is an important second messenger which can be generated in response to signals binding to stimulatory G-protein coupled receptors. Inside the cell, protein kinase A is a well-known cAMP sensor. Within the present study, cAMP-dependent changes in the phosphorylation (phospho) pattern of lysosomal membrane and lysosome-associated proteins were investigated. The serine phospho-site at position 133 (S133) of the lysosome-associated SNARE-associated protein (SNAPIN) was found to be significantly downregulated in the phosphoproteomic data set of the lysosomal membrane fraction after elevating the intracellular cAMP level by a Forskolin/dibutyryl-cAMP (FSK/db-cAMP) treatment. In addition to that, a tendency of SNAPIN to dissociate from the lysosomes upon elevated cAMP levels was observed. Therefore, the cAMP-dependent downregulation of the S133 phospho-site detected in the MS-based approach could have also been caused by a translocation of SNAPIN after FSK/db-cAMP treatment. Nevertheless, a dephosphorylation of SNAPIN was observed in 2D-gelelectrophoresis of whole cell protein lysate after elevating the intracellular cAMP level. This observation confirmed the findings of the phosphoproteomic study although the dephosphorylation site could not be mapped to a specific amino acid position by 2D-gelelectrophoresis. Pull-down assays with a phosphomimetic proteoform of the S133 phospho-site (S133E) indicate an interaction of the biogenesis of lysosome-related organelles complex-1 subunit 6 (BLOC1S6) with the S133E but not the S133A SNAPIN isoform (serine to alanine substitution mimics unphosphorylated SNAPIN at position 133). Furthermore, SNAPIN knock-out (KO) was associated with decreased endocytosis and increased exocytosis of lysosomal hydrolases compared to wildtype cells. Elevation of intracellular cAMP levels neither influenced these processes in wildtype nor in SNAPIN KO cells. This indicates that the dephosphorylation of SNAPIN detected after FSK/db-cAMP treatment might not be involved in regulating these secretion or uptake events.<br /> Posttranslational modifications (PTMs) of lysine and arginine residues are important regulators in different cellular processes. In bottom-up mass spectrometry-based approaches, these PTMs are most commonly enriched with pan-specific antibodies. Here, the investigations are limited to one specific PTM and depend on the quality and availability of proper antibodies in order to obtain an efficient enrichment of modified peptides. In addition to that, huge amounts of samples are usually required in these immunoprecipitation approaches which might be problematic when dealing with limited amounts of starting material. The present study aimed at developing and testing of two novel antibody-free techniques for improving the detection of specific PTMs from small quantities of starting sample. For example, such techniques would facilitate PTM analysis of enriched lysosomes or other purified subcellular compartments. The first technique, called the serial digestion workflow, was assumed to enhance the identification of lysine modifications. Compared to that, the second method, called the Nα-selective derivatization workflow (derivatization of primary amine group at amino-terminus), was hypothesized to improve the detection of PTMs occurring on both, lysine and arginine residues. Applying the two workflows to proteins from human embryonic kidney (HEK 293) cell lysate was not associated with an increased identification of lysine modifications. In contrast, sample treatment according to the Nα-selective derivatization method yielded an increase in the detection rate of arginine methylation. However, a similar rise was observed in both workflows after strong cation exchange chromatography which was originally performed as a sample clean-up procedure. Here, depending on the sample type, either the identification of arginine or of lysine and arginine methylation was increased. Comparison of the methylated peptides detected in the different samples showed only a partial overlap. This indicates the necessity of analyzing different samples coming from both workflows in order to get the most comprehensive protein methylation data set. Based on these findings, a new hybrid workflow is suggested.