Membrane targeting with palmitoylated lysine added to PP1‐disrupting peptide induces PP1‐independent signaling

Protein phosphatase‐1 (PP1) is a ubiquitous enzyme involved in multiple processes inside cells. PP1‐disrupting peptides (PDPs) are chemical tools that selectively bind to PP1 and release its activity. To restrict the activity of PDPs to a cellular compartment, we developed PDP‐Mem, a cell membrane‐targeting PDP. The membrane localization was achieved through the introduction of a palmitoylated lysine. PDP‐Mem was shown to activate PP1α in vitro and to localize to the membrane of HeLa Kyoto and U2OS cells. However, in cells, the combination of the polybasic sequence for cell penetration and the membrane targeting palmitoylated lysine activates the MAPK signaling pathway and induces cytoplasmic calcium release independently of PP1 activation. Therefore, when targeting peptides to cellular membranes, undesired effects induced by the targeting sequence and lipid modification need to be considered.

allow a more selective dephosphorylation of substrates and regulation of signaling. Here, we report the development of PDP-Mem, which targets the PP1 activating sequence to the plasma membrane. In vitro, PDP-Mem activates PP1α, whereas its inactive form PDPm-Mem shows little activation. However, in cells, also the inactive peptide induces a calcium release into the cytoplasm (calcium spiking) and leads to the phosphorylation of MEK1/2. By testing a number of polybasic sequences with a palmitoylated lysine, we show here that the combination of the polybasic sequence for cell penetration and the palmitoylated lysine employed for cell membrane targeting induces signaling in cells independently of the PP1 activation.  (Thermo Fisher Scientific) and 1% penicillin/streptomycin (P/S) (Sigma-Aldrich). Cells were maintained at 37 C in a fully humidified atmosphere containing 5% CO 2 .

| Peptide synthesis
Peptides were synthesized following previously reported solid-phase peptide synthetic (SPPS) strategies. 10 For further details, see Supporting Information.

| PP1 deinhibition assay
The PP1 deinhibition assay was performed following previously published protocols. 11   The cells were then scraped off and centrifuged for 10 min at 4 C and 14 000 rpm. The supernatant was mixed with 2Â SB (125 mM Tris pH 6.8, 20% glycerol, 4% SDS, 0.02% bromophenol blue, 5% β-mercapoethanol) and boiled for 5-10 min at 95 C. SDS-PAGE and western blot methods are described in the Supporting Information.

| RESULTS
Palmitoylation is the post-translational addition of palmitic acid (16 carbon fatty acid) to cysteine either through a reversible coupling to the thiol side chain or irreversibly to the N-terminal amine group and ε-amino group of lysine. [14][15][16] It has long been known to be involved with protein trafficking to and from the cellular membrane, as it greatly increases the hydrophobicity of proteins and peptides. 17 The addition of a palmitoyl group to a peptide was shown to enhance its interaction with lipid bilayers and can facilitate the cellular uptake. [18][19][20][21][22][23] Thus, palmitoylation was selected in order to impart a membrane-associating functionality to the PDPs, by incorporating palmitic acid into the PDP-Nal sequence, the most recently optimized PDP. 24  From the fluorescence microscopy study, it cannot be concluded whether PDP-Mem is localized on the outside of the cell, at the inner leaflet of the plasma membrane pointing the active sequence inside the cytoplasm, or retained within the membrane. Therefore, we sought to investigate whether PDP-Mem activates PP1 not only in vitro but also in intact cells. Few membrane proteins such as the TGFβI receptor are known to be dephosphorylated by PP1. 25 As no antibodies for those specific sites are available, we tested the effect on the calcium release by the cells into the cytoplasm upon PDP-Mem treatment. This approach was already used to characterize the activity of other PDPs. 26 HeLa cells were stained with Fluo-4 to monitor the calcium release. 27 The previous PDP versions containing the RVxF motif induced a repeated calcium spiking of the cells by activating PP1, whereas the inactive forms showed no spiking. 26 The PDPs were taken up by the cells quickly showing a rapid spiking response. 26 Here, the addition of PDP-Mem led to a single spiking event from all cells right after the treatment and no repeated spiking over a longer time ( Figures 4A and S8). The rapid onset of the spike can be explained by  (Table S2, Figures S8-S11).
a very quick association of the peptide with the cell membrane. If the observed spiking is induced by the direct activation of PP1 by PDP-Mem, the results suggest a quick flipping of the peptide to the inner leaflet. When removing the Ca 2+ from the imaging buffer, the same results were obtained (Figures 4B and S10). This suggests that the calcium influx does not or only partially originate from calcium contained in the medium. It therefore appears that the flipping of the peptides from the extra-to the intra-cellular side of the membrane does not damage the membrane leading to a calcium influx. When adding PDPm-Mem to the HeLa cells, the results were not consistent and reproducible, with and without the presence of external calcium ( Figures 4B, S9, and S11). Spiking events were recorded in more than half the repetitions even though the inactive RATA form should not activate PP1. These results suggest that the peptides have an additional effect on the cells, which is independent of the PP1 activation.
PP1 activation was shown to influence MAPK signaling. 24,28 Therefore, as a different approach to test the capacity of PDP-Mem to activate PP1, we looked at its effect on the phosphorylation of MEK1/2. U2OS cells were treated for 10 min (within the time where PDP-Mem was located at the plasma membrane, Figure 2) with PDP-Nal, PDPm-Nal, PDP-Mem, PDPm-Mem, and Mem-1 ( Table 2) PDPm-Nal had no effect. 24 In previous publications, where peptides were targeted to cellular membranes, the palmitoyl group and the polybasic sequence were not combined this closely (with K(Pal) included into the polybasic sequence). 19,21,23 The C-terminus of the PDP needs to be free to activate PP1. Therefore, K(Pal) cannot be moved there to create more distance to the polybasic sequence. As another option, we  (Table 2). For all those short peptides, an increase in MEK1/2 phosphorylation was observed after a 10 min treatment of the cells ( Figure 5B). For the 3 sequences that contain an N-terminal K(Pal), the effect is even more pronounced than for Mem-1. They are therefore not an option to target the PDP to the cellular membrane.

| CONCLUSION
In summary, we have designed and synthesized a membrane targeted PP1 activator. We successfully incorporated a palmitoylation onto a lysine in PDP-Mem, which imparts a temporary cell membrane localization property to the peptide. As seen from the PP1 deinhibition assay, addition of this post-translational modification only slightly reduces the activating ability of the PDP compared with the template peptide, PDP-Nal. 24 Also, mutation of the RVTF motif to RATA leads to a strong reduction in this activation efficacy. The activity assays of PDP-Mem and PDPm-Mem in intact cells did not yield the expected results. As PDPm-Mem did not activate PP1 in vitro and PDPm-Nal showed no effect on cells, 24 we expected PDPm-Mem to have no effect on intact cells either. Yet, we observed that it can induce calcium spiking and also leads to an increase of MEK1/2 phosphorylation. As PDP-Mem and PDPm-Mem showed similar effects in cells, the K(Pal) combined with the polybasic sequence might induce PP1 unrelated cell signaling. Short peptides consisting only of a polybasic sequence and the K(Pal) in different combinations were used to test this hypothesis. All of them lead to an increase in MEK1/2 phosphorylation. The results showed that the polybasic sequence combined with K(Pal) is sufficient to induce signaling. Therefore, a different method has to be used to target the PP1 activating sequence to cell membranes. When using K(Pal) to target peptides to cellular membranes, the effect on MAPK and other signaling pathways should be tested, to exclude any effect other than the desired one. Based on these results, further development and optimization of PDPs for compartment-specific targeting are required to enable a more precise spatial, and temporal, 29 control of PP1 activation.