The dual-specificity phosphatase JSP1, and its relative DUSP15, are unique among members of the PTP family in that they contain a potential myristoylation consensus sequence at the N-terminus (MG
NGMXK). In their study of VHY/DUSP15, Mustelin’s group demonstrated that the VHY and VHX proteins incorporated 14
C in cells metabolically labeled with [14
C]-myristic acid [29
]. The goal of the present study was to demonstrate directly that JSP1 was myristoylated, to apply a mass spectrometric approach to identify the residue in JSP1 that was modified, and to analyze whether myristoylation had an effect on JSP1 function.
Modification by covalently linked fatty acids, i.e. myristoylation or palmitoylation, has been shown to occur on a wide variety of signaling proteins. These hydrophobic modifications can confer reversible association with membranes and other signaling proteins, which modulates the specificity and efficiency of signal transduction [30
]. N-myristoylation is the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine of eukaryotic and viral proteins. The process is catalyzed by N-myristoyl transferase (NMT), and generally occurs co-translationally following removal of the initiator methionine residue by methionylaminopeptidases. The consensus sequence for NMT protein substrates is Met-Gly-X3
-Ser/Thr-Lys/Arg-, but only the requirement for Gly at the N-terminus is absolute. For example, the tyrosine kinase c-Abl, a myristoylated protein, contains Gly and Lys at positions 2 and 7, respectively, but no Ser/Thr at position 6 [31
Between 0.5% and 3% of all eukaryotic proteins are N-myristoylated. These proteins have a broad range of functions and include protein kinases and phosphatases, Gα proteins, nitric oxide synthase (NOS), ADP-ribosylation factors (ARFs), and membrane-or cytoskeleton-associated structural proteins (e.g. MARCKS). The myristoyl moiety serves several functions: it can promote reversible binding and localization to membranes, stabilize the conformation of proteins, and regulate protein interactions. For example, myristoylation of Src is required for its localization to the plasma membrane, which is critically important for its proper function. A non-myristoylated mutant of Src, although catalytically active, has no transforming activity [33
]. Stabilization of a protein by myristoylation is exemplified by the example of cAMP-dependent protein kinase, where the myristoyl group binds to a hydrophobic cleft in the protein, thus stabilizing its tertiary strucutre [35
]. An unusual example for the regulation of protein interaction is NADH-cytochrome b5 reductase (b5R), where myristoylation interferes with binding of the signal recognition particle, resulting in a part of b5R escaping the ER insertion pathway and relocating to the outer mitochondrial membrane [36
]. Myristoylation has also been implicated in the regulation of apoptosis. Although normally a co-translational process, several proteins, including the proapoptotic protein Bid, actin and the Ser/Thr kinase Pak2, become myristoylated at newly generated N-terminal glycines after caspase cleavage [32
]. In the case of Bid, the myristoylated fragment relocates to the mitochondrial membrane, where it induces oligomerization of Bak and subsequent cytochrome c release [37
Myristoylation can also influence the movement and final destination of a signaling protein within the cell. We observed that myristoylation of JSP1 determined its localization to distinct sites in the cytoplasm. Signaling from internal membranes is now considered to be an important aspect of the spatial and temporal regulation of signaling pathways, e.g. the Ras/MAPK pathway [38
]. In order to specify the JSP1-containing structures, we tested colocalization of JSP1 with various marker proteins. Although we found that JSP1 colocalized with Golgi markers, further study is required to ascertain more precisely the distribution of JSP1 within the cell and to define its phosphorylated substrates and, thereby, its mechanism of action.
We have previously reported that JSP1 specifically activated the JNK pathway, hence the name JNK-stimulatory phosphatase 1 [20
]. This result was supported by a second study that showed that the murine DSP JNK-pathway associated phosphatase (JKAP), a splice isoform of JSP1, specifically activated JNK when overexpressed in human embryonic kidney 293T cells [21
]. Overexpression of a catalytically inactive mutant (JKAP-C88S) blocked tumor necrosis factor-α-induced JNK activation. Moreover, in murine JKAP−/−
embryonic stem cells, JNK activation was abolished in response to tumor necrosis factor-α and transforming growth factor-β, but not in response to ultraviolet-C irradiation. These data illustrate that JSP1 is required for cytokine-induced activation of the JNK pathway. In contrast, Ayoama et al.
suggested that when overexpressed in Cos-7 cells, JSP1/LMW-DSP2 dephosphorylated and inactivated p38, and, to a lesser extent, JNK after stimulation of the kinases with the appropriate agonists [22
]. In addition, Alonso et al.
reported a negative effect of JSP1/VHX on T cell receptor-induced activation of ERK2 in transfected Jurkat T cells [23
]. The reason for these discrepancies is unclear, but could be due to differences of JSP1 function in the different cell systems used. In the present study, we have confirmed activation of JNK and its downstream transcription factor c-JUN by JSP1, which was dependent on a functional myristoylation site. Since myristoylation was not necessary for the intrinsic phosphatase activity of JSP1, but determined the subcellular localization of the phosphatase, this result suggests that correct localization of JSP1 to specific subcellular compartments is critically important for its functional activity in the JNK signaling pathway.
Overexpression of wild type JSP1, but neither the myristoylation-deficient mutant nor a catalytically inactive mutant, induced ~ 30% of the transfected cells to float off the dish and undergo apoptosis. Interestingly, the cells could tolerate high levels of the myristoylation-deficient mutant and remain attached, whereas similar levels of the wild type protein induced apoptosis. This study illustrates that the toxicity of wild type JSP1 presents a technical challenge that prohibits functional analysis using overexpression systems. Consistent with this, we have not been able to create stable cell lines expressing JSP1-wt constitutively, and almost all of the existing cell lines we have examined do not express detectable levels of JSP1 protein. In fact, in order to generate sufficient quantities of wild type protein for mass spectrometric analysis of the myristoylation site, we used 293T cells as expression system, since the presence of the SV40 large T antigen in these cells enhances their resistance to apoptosis.
Apoptosis is a tightly regulated mechanism for disposal of damaged cells and to remove cells during normal growth and development [27
]. Cells that undergo apoptosis initially become rounded, which is accompanied or followed by membrane blebbing, resulting in small vesicles termed apoptotic bodies. Inside the cell, apoptosis is characterized by condensation and fragmentation of the nucleus [27
], as well as hydrolysis of nuclear DNA into distinct fragments by endonucleases [41
]. Two main pathways lead to caspase-dependent apoptosis. In the extrinsic pathway, binding of death ligands to their respective receptors recruit adaptor proteins, such as Fas-associated death domain protein (FADD), which in turn bind and aggregate caspase-8 molecules, resulting in their autocleavage and activation. Active caspase-8 proteolytically processes and activates downstream caspases, eventually leading to substrate proteolysis, such as the nuclear poly (ADP-ribose) polymerase PARP [40
]. In the intrinsic pathway, cell stress or damage activates members of the pro-apoptotic BH3-only protein family, which induce permeabilization of the outer mitochondrial membrane. Release of mitochondrial cytochrome c triggers assembly of a caspase-9-activating complex and subsequent activation of the downstream caspase cascade. These pathways are not mutually exclusive and are connected by caspase-8, which can trigger proteolysis of the BH3-only protein BID. When we analyzed the phenotype of JSP1-transfected, floating cells, we observed typical signs of apoptotic cell death, including condensed chromatin in the nucleus. Further analysis revealed that floating could be inhibited by treating the cells with a pan-caspase inhibitor, Z-VAD-FMK, simultaneously with JSP1 transfection. Induction of apoptosis was further implicated by the presence of cleaved Caspase-9 and PARP in floating cells (with high expression of wild type JSP1), but not in attached cells (low JSP1 expression), or cells with equally high expression of the myristoylation mutant. These results suggest that JSP1 induces apoptosis when overexpressed in cells, and further demonstrate the importance of myristoylation for this functional activity of JSP1.
It is reasonable to suggest that elevated JNK activity may precede the detachment and induction of apoptosis in the sub-population of cells expressing high levels of JSP1. We attempted to test this by treating cells with the JNK-inhibitor SP600125 concomitant with transfection, to determine whether inhibition of JNK abrogated the effect despite JSP1 expression. However, these efforts were frustrated by the lack of specificity of SP600125, which has also been reported by others [42
]. Resolution of the importance of JNK activation will require further experimentation.
In summary, JSP1, and VHY/DUSP15, are unique among the members of the PTP family in having a putative N-terminal myristoylation sequence and unusual in light of their potential to promote signaling. In this study, we demonstrate that JSP1 is myristoylated. Although this modification is not required for the intrinsic phosphatase activity of JSP1, we demonstrate that myristoylation is necessary for the ability of JSP1 to activate JNK signaling and to trigger apoptosis upon overexpression in our cell models. Further studies will focus on identification of physiological substrates of JSP1, to reveal the mechanism underlying its effects on JNK signaling and whether this is linked to the observed triggering of apoptosis.