The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway.
Title: The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus Justa-Schuch D, Moller U, Geiss-Friedlander R Ref: Cell Mol Life Sciences, 71:3611, 2014 : PubMed
The intracellular prolyl peptidase DPP9 is implied to be involved in various cellular pathways including amino acid recycling, antigen maturation, cellular homeostasis, and viability. Interestingly, the major RNA transcript of DPP9 contains two possible translation initiation sites, which could potentially generate a longer (892 aa) and a shorter version (863 aa) of DPP9. Although the endogenous expression of the shorter DPP9 form has been previously verified, it is unknown whether the longer version is expressed, and what is its biological significance. By developing specific antibodies against the amino-terminal extension of the putative DPP9-long form, we demonstrate for the first time the endogenous expression of this longer isoform within cells. Furthermore, we show that DPP9-long represents a significant fraction of total DPP9 in cells, under steady-state conditions. Using biochemical cell fractionation assays in combination with immunofluorescence studies, we find the two isoforms localize to separate subcellular compartments. Whereas DPP9-short is present in the cytosol, DPP9-long localizes preferentially to the nucleus. This differential localization is attributed to a classical monopartite nuclear localization signal (K(K/R)X(K/R)) in the N-terminal extension of DPP9-long. Furthermore, we detect prolyl peptidase activity in nuclear fractions, which can be inhibited by specific DPP8/9 inhibitors. In conclusion, a considerable fraction of DPP9, which was previously considered as a purely cytosolic peptidase, localizes to the nucleus and is active there, raising the intriguing possibility that the longer DPP9 isoform may regulate the activity or stability of nuclear proteins, such as transcription factors.
Sumoylation affects many cellular processes by regulating the interactions of modified targets with downstream effectors. Here we identified the cytosolic dipeptidyl peptidase 9 (DPP9) as a SUMO1 interacting protein. Surprisingly, DPP9 binds to SUMO1 independent of the well known SUMO interacting motif, but instead interacts with a loop involving Glu(67) of SUMO1. Intriguingly, DPP9 selectively associates with SUMO1 and not SUMO2, due to a more positive charge in the SUMO1-loop. We mapped the SUMO-binding site of DPP9 to an extended arm structure, predicted to directly flank the substrate entry site. Importantly, whereas mutants in the SUMO1-binding arm are less active compared with wild-type DPP9, SUMO1 stimulates DPP9 activity. Consistent with this, silencing of SUMO1 leads to a reduced cytosolic prolyl-peptidase activity. Taken together, these results suggest that SUMO1, or more likely, a sumoylated protein, acts as an allosteric regulator of DPP9.
Protein degradation is an essential process that continuously takes place in all living cells. Regulated degradation of most cellular proteins is initiated by proteasomes, which produce peptides of varying length. These peptides are rapidly cleaved to single amino acids by cytoplasmic peptidases. Proline-containing peptides pose a specific problem due to structural constrains imposed by the pyrrolidine ring that prevents most peptidases from cleavage. Here we show that DPP9, a poorly characterized cytoplasmic prolyl-peptidase, is rate-limiting for destruction of proline-containing substrates both in cell extracts and in intact cells. We identified the first natural substrate for DPP9, the RU1(34-42) antigenic peptide (VPYGSFKHV). RU1(34-42) is degraded in vitro by DPP9, and down-regulation of DPP9 in intact cells results in increased presentation of this antigen. Together our findings demonstrate an important role for DPP9 in peptide turnover and antigen presentation.
Stimulation of phospholipase D (PLD) in HEK-293 cells expressing the M(3) muscarinic receptor by phorbol ester-activated protein kinase C (PKC) apparently involves Ral GTPases. We report here that PKC, but not muscarinic receptor-induced PLD stimulation in these cells, is strongly and specifically reduced by expression of dominant-negative RalA, G26A RalA, as well as dominant-negative Ras, S17N Ras. In contrast, overexpression of the Ras-activated Ral-specific guanine nucleotide exchange factor, Ral-GDS, specifically enhanced PKC-induced PLD stimulation. Moreover, recombinant Ral-GDS potentiated Ral-dependent PKC-induced PLD stimulation in membranes. Epidermal growth factor, platelet-derived growth factor, and insulin, ligands for receptor tyrosine kinases (RTKs) endogenously expressed in HEK-293 cells, apparently use the PKC- and Ras/Ral-dependent pathway for PLD stimulation. First, PLD stimulation by the RTK agonists was prevented by PKC inhibition and PKC down-regulation. Second, expression of dominant-negative RalA and Ras mutants strongly reduced RTK-induced PLD stimulation. Third, overexpression of Ral-GDS largely potentiated PLD stimulation by the RTK agonists. Finally, using the Ral binding domain of the Ral effector RLIP as an activation-specific probe for Ral proteins, it is demonstrated that endogenous RalA is activated by phorbol ester and RTK agonists. Taken together, strong evidence is provided that RTK-induced PLD stimulation in HEK-293 cells is mediated by PKC and a Ras/Ral signaling cascade.
