5. Results
5.1. Investigating cAMP mediated phosphorylations of lysosomal and lysosome-associated
5.1.2. Identification of significantly regulated phosphopeptides upon elevated
5.1.2.2. Phosphopeptide enrichment from lysosomal fractions
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are listed in table 5.4. Besides that, 18 phosphopeptides were detected in at least two DMSO treated samples but not in any FSK/db-cAMP replicate, thus belonging to the highly downregulated peptide population (Table 5.6). The highly upregulated phospho-site group comprises peptides found in at least two FSK/db-cAMP replicates but not in any DMSO control.
Within the present study, 23 phosphopeptides were found to be highly upregulated upon elevated intracellular cAMP levels (Table 5.5).
Known PKA target sites of the CD44 antigen (S697) and the CAD protein (S1406) were identified as being upregulated in the lysosomal membrane fraction after FSK/db-cAMP treatment (PhosphoSitePlus online repository; phosphosite.org, retrieved 09.05.2019; note that the S697 CD44 phospho-site corresponds to the S316 site described by Tzircotis et al. (2006) (Carrey et al.
1985)). Here, an increase in the protein level was not detected. The upregulated phospho-site of the Vesicle-trafficking protein SEC22b, identified after phosphopeptide enrichment from the whole cell digest (Table 5.1), was also found as being significantly upregulated in the lysosomal membrane fractions (Figure 5.3, table 5.4). This demonstrates the comparability of the stimulus applied to the cells within the different experiments, although the starting material for phosphopeptide enrichment was different. The presence of non-lysosomal proteins among the regulated phosphopeptides in the membrane fraction indicate a certain impurity of the lysosome preparation. Highly or significantly regulated phospho-sites were identified for the lysosomal and lysosome-associated proteins regulatory-associated protein of mTOR (RPTOR), serine/threonine-protein kinase mTOR, run domain Beclin-1-interacting and cysteine-rich domain-containing protein (RUBCN), Mucolipin 1, and the SNARE-associated protein (SNAPIN).
The protein levels of RPTOR, mTOR and Mucolipin 1 were not regulated in the lysosomal membrane fraction after FSK/db-cAMP treatment. Compared to that, RUBCN and SNAPIN were not detected in the non-phospho enriched samples thus hindering conclusions about the cAMP-induced changes in protein levels within this lysosomal fraction. The phospho-sites identified for the lysosomal proteins are not assigned as PKA target sites in the PhophoSitePlus online repository (phosphosite.org, retrieved 09.05.2019). Compared to that, phosphorylation of SNAPIN (at S50) and Mucolipin 1 (at S557 and S559) have already been associated with PKA activity (Chheda et al. 2001; Vergarajauregui et al. 2008). Interestingly, the S133 phospho-site of SNAPIN identified within the present study was downregulated after FSK/db-cAMP treatment.
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Figure 5.3: Phosphopeptide enrichment from lysosomal membrane fraction.
Lysosomes from isotopically labeled cells were enriched and subsequently separated into a water soluble and membrane fraction. After tryptic digestion, phosphopeptide enrichment was performed by TiO2. The experiment was performed in three biological replicates. Phosphopeptides derived from the membrane fractions were analyzed with an Orbitrap Fusion Lumos mass spectrometer. Raw data were searched against a human reference database by using the Mascot search engine. Statistical analysis (FSK/db-cAMP vs DMSO) was performed on phosphopeptides found in at least two out of three replicates (adjusted p-value threshold 0.05) by using the limma package implemented in an R-script. Significantly regulated phosphopeptides (adjusted p-value <0.05 and log2 fold change <1 or >1) are highlighted as red points.
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Table 5.4: Significantly regulated phosphopeptides detected in the lysosomal membrane fraction after FSK/db-cAMP treatment.
List of proteins corresponding to the twelve significantly regulated phosphopeptides (red points in figure 5.3) identified after statistical analysis of the phospho-data set derived from the lysosomal membrane fraction (FSK/db-cAMP vs. DMSO). The ratios of the corresponding protein abundance (FSK/db-(FSK/db-cAMP vs. control) determined by MS analysis of the non-phopho-enriched samples are shown.
Uniprot accession
Protein name Log2 fold change
Adj. p-value Protein ratio (FSK/db-cAMP vs.
DMSO) P52594 Arf-GAP domain and FG
repeat-containing protein 1
-2.7 0.0015 Not found
O95295 SNARE-associated protein SNAPIN
-1.8 0.0126 Not found
Q4KMP7 TBC1 domain family member 10B
-1.8 0.0097 1.12
O43521 Bcl-2-like protein 11 1.6 0.0161 Not found
P13591 Neural cell adhesion molecule 1
2.5 0.0126 1.04
O94887 FERM, ARHGEF and pleckstrin domain-containing protein 2
2.8 0.0044 0.47
Q9GZU1 Mucolipin 1 3.8 0.0002 1.14
P07197 Neurofilament medium polypeptide
4.0 0.0002 0.97
O75396 Vesicle-trafficking protein SEC22b
4.2 0.0036 Found in less than 2 replicates
Q7L9L4 MOB kinase activator 1B 2.7 0.0216 Not found
P62753 40S ribosomal protein S6 2.9 0.0389 0.81
Q9H2J7 Sodium-dependent neutral amino acid transporter
B(0)AT2
1.8 0.0307 0.93
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Table 5.5: Highly upregulated phosphopeptides detected in the lysosomal membrane fraction upon elevation of the intracellular cAMP level.
List of phosphopeptides found to be highly upregulated in the lysosomal membrane faction after FSK/db-cAMP treatment (found in at least two out of three FSK/db-cAMP replicates but not in any DMSO sample). For each phosphopeptide, protein abundance ratios determined in the non-phospho-enriched samples are listed.
Uniprot accession
Modified sequence Protein name Protein ratio
(FSK/db-cAMP vs. DMSO)
Q12846 AIEPQKEEADENYNSVNTR_1xPhospho_[S15(100)] Syntaxin-4 1.21
P54105 FEEESKEPVADEEEEDSDDDVEPITEFR_1xPhospho_[S17(100)] Methylosome subunit pICln 0.71
Q8N122 GVHIHQAGGSPPASSTSSSSLTNDVAKQPVSR_1xPhospho_[S10(100)] Regulatory-associated protein of mTOR 1.01
P55011 KESKGPIVPLNVADQK_1xPhospho_[S3(100)] Solute carrier family 12 member 2 0.82
Q9BQG0 KGVLGKSPLSALAR_1xPhospho_[S7(100)] Myb-binding protein 1A 0.82
Q8N7R7 KYSSCSTIFLDDSTVSQPNLR_1xPhospho_[Y/S/T] Cyclin-Y-like protein 1 Not found
Q8N7R7 KYSSCSTIFLDDSTVSQPNLR_1xPhospho_[S/T/Y] Cyclin-Y-like protein 1 Not found
P16070 LVINSGNGAVEDRKPSGLNGEASK_1xPhospho_[S16(100)] CD44 antigen 1.31
O95297 SESVVYADIR_1xPhospho_[Y6(100)] Myelin protein zero-like protein 1 1.16
P51610 ASAVSPANLPAVLLQPR_1xPhospho_[S5(100)] Host cell factor 1 Not found
Q5VUB5 DQSTSMSHINLLFSR_1xPhospho_[T/S] Protein FAM171A1 Not found
Q14789 KFSDAIQSKEEEIR_1xPhospho_[S3(100)] Golgin subfamily B member 1 1.00
P13746 KGGSYTQAASSDSAQGSDVSLTACKV_1xPhospho_[S20(99.3)] HLA class I histocompatibility antigen, A-11 alpha chain
0.94
Q9C0C9 KKSIPLSIK_1xPhospho_[S3(100)] E2 ubiquitin-conjugating enzyme Not found
P49792 KQSLPATSIPTPASFK_1xPhospho_[S3(100)] E3 SUMO-protein ligase RanBP2 0.90
Q9P2M7 KVSLVLEK_1xPhospho_[S3(100)] Cingulin 1.23
P04920 NISAGSLGSLLGHHHGQGAESDPHVTEPLMGGVPETR_1xPhospho_[S3(100)] Anion exchange protein 2 1.00
Q9Y2J2 RASALIDRPAPYFER_1xPhospho_[S3(100)] Band 4.1-like protein 3 0.76
O95613 RESEVLDLKEQLEK_1xPhospho_[S3(100)] Pericentrin 1.17
Q96II8 RISHEGSPVKPVAIR_1xPhospho_[S3(100)] Leucine-rich repeat and calponin
homology domain-containing protein 3
0.81
P27708 RLSSFVTK_1xPhospho_[S3(100)] CAD protein 1.14
Q86W92 RRPSDENTIAPSEVQK_1xPhospho_[S4(100)] Liprin-beta-1 Not found
P42345 TRTDSYSAGQSVEILDGVELGEPAHKK_1xPhospho_[S5(98.4)] Serine/threonine-protein kinase mTOR 0.97
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Table 5.6: Highly downregulated phosphopeptides detected in the lysosomal membrane fraction upon elevation of the intracellular cAMP level.
List of phosphopeptides found to be highly downregulated in the lysosomal membrane faction after FSK/db-cAMP treatment (found in at least two out of three DMSO replicates but not in any FSK/db-cAMP sample). For each phosphopeptide, protein abundance ratios determined in the non-phospho-enriched samples are listed.
Uniprot accession
Modified sequence Protein name Protein ratio
(FSK/db-cAMP vs. DMSO)
Q9BY89 AKLDPEPEKAAESPSPR_1xPhospho_[S15(99.5)] Uncharacterized protein KIAA1671 Not found
P19634 IPSAVSTVSMQNIHPK_1xPhospho_[S9(100)] Sodium/hydrogen exchanger 1 Not found
Q9H2J7 QSGSPTLDTAPNGR_1xPhospho_[S4(100)] Sodium-dependent neutral amino acid
transporter B(0)AT2
0.93 O15439 SGIDFGSLLKKDNEESEQPPVPGTPTLR_1xPhospho_[T24(100)] Multidrug resistance-associated protein 4 0.54 P52594 SLLGDSAPTLHLNKGTPSQSPVVGR_2xPhospho_[T16(100);_S20(100)] Arf-GAP domain and FG
repeat-containing protein 1
Not found
P52594 SLLGDSAPTLHLNKGTPSQSPVVGR_2xPhospho_[T/S] Arf-GAP domain and FG
repeat-containing protein 1
Not found Q09666 TVIRLPSGSGAASPTGSAVDIR_1xPhospho_[S13(100)] Neuroblast differentiation-associated
protein AHNAK
1.04
Q12933 AAASVTPPGSLELLQPGFSK_1xPhospho_[S4(99.2)] TNF receptor-associated factor 2 0.90
O75427 AAAVAAPLAAGGEEAAATTSVPGSPGLPGRR_1xPhospho_[T/S] Leucine-rich repeat and calponin homology domain-containing protein 4
Not found P20042 SGDEMIFDPTMSKK_1xPhospho_[S1(100)] Eukaryotic translation initiation factor 2
subunit 2
Not found Q5T1M5 DSGSDGHSVSSRDSAAPSPIPGADNLSADPVVSPPTSIPFK_2xPhospho_[S18(100);_S/T] FK506-binding protein 15 0.87
Q9BXB5 HLSVGAPGVVTITHHKSPAAAR_1xPhospho_[S17(100)] Oxysterol-binding protein-related protein 10
Not found
Q9H1B7 KASPEPPDSAEGALK_1xPhospho_[S3(100)] Probable E3 ubiquitin-protein ligase
IRF2BPL
Not found
Q92622 KHESPLLVTK_1xPhospho_[S4(100)] Run domain Beclin-1-interacting and
cysteine-rich domain-containing protein
Not found
Q2NKX8 RFPEAEALSPEQAAHYLR_1xPhospho_[S9(100)] DNA excision repair protein ERCC-6-like 0.71
Q4KMP7 RHGAPAAPSPPPR_1xPhospho_[S9(100)] TBC1 domain family member 10B 1.13
Q13427 RSETPPHWR_1xPhospho_[T4(99.6)] Peptidyl-prolyl cis-trans isomerase G Not found
Q9BXB4 SFSLASSSNSPISQR_1xPhospho_[S10(99.6)] Oxysterol-binding protein-related protein 11
0.92
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The current literature suggests important role of SNAPIN in the proper lysosome functionality (see section 2.5). Therefore, the present study aimed at further elucidating the importance of SNAPIN and the identified downregulated phospho-site at S133 in different processes such as the internalization and secretion of acidic hydrolases as well as lysosome positioning.