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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Pept Sci. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2922070

Design of a bioactive cell-penetrating, peptide: when a transduction domain does more than transduce


The discovery of cell-penetrating peptides (CPPs) has facilitated delivery of peptides into cells to affect cellular behavior. Previously, we were successful at developing a phosphopeptide mimetic of the small heat shock-like protein HSP20 [1, 2]. Building on this success we developed a cell-permeant peptide inhibitor of mitogen-activated protein kinase-activated protein kinase 2 (MK2) [3]. It is well documented that inhibition of MK2 may be beneficial for a myriad of human diseases including those involving inflammation and fibrosis. During the optimization of the activity and specificity of the MK2 inhibitor (MK2i) we closely examined the affect of cell-penetrating peptide identity. Surprisingly, the identity of the CPP dictated kinase specificity and functional activity to an extent that rivaled that of the therapeutic peptide. The results reported herein have wide implications for delivering therapeutics with CPPs and indicate that judicious choice of CPP is crucial to the ultimate therapeutic success.

Keywords: mitogen-activated protein kinase-activated protein kinase 2, cell-penetrating peptide, inhibitor specificity, peptide therapeutics


Over the past 20 plus years since the simultaneous discovery by Frankel and Pabo [4] and by Green and Loewenstein [5] that the TAT protein from the HIV virus could be taken up by cells, a tremendous body of work investigating the mechanism and limitations of the uptake has been generated [69]. In addition, several investigators have identified new sequences that act as CPPs [3, 1013] (commonly used CPPs also listed in [14]). The mechanism of entry into the cell by these cell-permeating peptides is still an active topic of investigation [1416]. Less widely investigated is the effect of the identity or primary structure of the CPP on the activity of the molecule to which it is attached.

Mitogen-activated protein kinase-activated protein kinase 2, MAPKAP-K2 (MK2), controls gene expression at both the transcriptional and post-transcriptional levels as well as cytoskeletal architecture [17]. Two MAP kinases, p38α and p38β, activate MK2 [18]. Environmental stresses including heat shock, hypoosmolarity, and hypoxia and inflammatory cytokines, such as TGF-β1, IL-1, TNF-α, IL-6, and GM-CSF, activate the p38/MK2 pathway [1926]. When activated, MK2 increases the translation and stability of cytokine mRNA and causes actin reorganization [19, 27, 28].

Inhibiting MK2 may be beneficial for a myriad of human diseases including endotoxic shock [29], pancreatitis [30], asthma [31], localized inflammatory disease [31], atherosclerotic cardiovascular disease [32], Alzheimer’s disease [33, 34], cancer [35], neural ischemia [34], rheumatoid arthritis [36], and inflammatory bowel disease [37]. Thus, an MK2 inhibitor may have an enormous impact on treating human disease. While several small molecule inhibitors of MK2 are under development, none has yet been approved by the United States FDA [38].

We previously reported on an MK2 inhibitor peptide, WLRRIKAWLRRIKALNRQLGVAA, [39] that was derived from the sequence published by Hayess and Benndorf [40]. However, the initial work with this peptide, while demonstrating functional activity, also demonstrated poor cell viability in vitro. This observation led us to explore the potential origin of toxicity.

Work by Lukas, et al. suggests that highly basic peptides can serve as inhibitors of myosin light chain kinase [41]. This information coupled with the apparent toxicity of the highly basic WLRRIKAWLRRIKALNRQLGVAA led us to hypothesize that basic cell penetrating peptides may lead to nonspecific kinase inhibition. To further investigate the impact of CPP on activity, alternate CPPs were examined. Two of these CPPs were based on HIV’s TAT protein --YGRKKRRQRRR and YARAAARQARA [4245]. The original CPP, WLRRIKAWLRRIKA, was also tested along with its non-functional monomer, WLRRIKA. Two additional, novel CPPs were introduced during this work, KAFAKLAARLYR and FAKLAARLYR. These two CPPs are based on the antithrombin III heparin-binding domain [46, 47]. We have shown that all of the CPPs tested in these studies enter cells (data not shown). Kinase inhibition was evaluated using radiometric assays to better identify how modifications to the CPP influenced MK2 inhibition and to better determine the specificity of complete (CPP and therapeutic domain coupled) peptides.

These studies suggest that care must be taken in choosing CPPs for the delivery of bioactive molecules into cells. The sequence of the CPP can influence the kinase inhibition activity and specificity. In addition, progress has been made toward designing a peptide-based inhibitor with improved potency and specificity for MK2. Future studies will address the biological activity of our novel MK2 inhibitor peptides in cell culture and in vivo.


Peptide synthesis and purification

Peptides were synthesized on Rink-amide or Knorr-amide resin (Synbiosci Corp.) using standard FMOC chemistry [48] on a Symphony® Peptide Synthesizer (Protein Technologies, Inc.). The coupling reagent for the amino acids (Synbiosci Corp.) was HBTU/NMM (Anaspec/Sigma). Following synthesis, the peptide was cleaved from the resin with a trifluoroacetic acid-based cocktail, precipitated in ether, and recovered by centrifugation. The recovered peptide was dried in vacuo, resuspended in MilliQ purified water, and purified using an FPLC (ÄKTA Explorer, GE Healthcare) equipped with a 22/250 C18 prep-scale column (Grace Davidson). An acetonitrile gradient with a constant concentration of either 0.1% trifluoroacetic acid or 0.1% acetic acid was used to achieve purification. Desired molecular weight was confirmed by time-of-flight MALDI mass spectrometry using a 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems).

Fluorescence-based kinase activity assay

The Omnia® Kinase Assay for MAPKAP-K2 kit (Invitrogen) was used to determine the reaction velocity for MK2 in the presence and absence of each of the peptides listed in Table 1. The kit contains a proprietary reaction buffer to which the following were added (final concentrations are given): 1 mM ATP, 0.2 mM DTT, 10 μM MAPKAP-K2 Sox-modified peptide substrate, 5 ng MK2, and the peptide inhibitor of interest (final volume of 50 μl). Human MK2 was purchased from Millipore. The reactions were performed at 30°C in the wells of a low-protein-binding 96-well plate provided with the kit, and fluorescence readings (excitation = 360 nm, emission = 485 nm) were taken every 30 seconds for 20 minutes using a SpectraMax M5 Spectrophotometer (Molecular Devices). Reaction velocity was determined for each reaction well from the slope of a plot of relative fluorescence units versus time. Each inhibitor peptide was tested at least at four concentrations, 12.5, 25, 50 and 100 μM in triplicate.

Table 1
Peptides tested in fluorescent-based kinase activity assay

Radiometric IC50 and kinase activity determination

A commercial radiometric assay service was used to test the specificity and potency of complete peptides (CPP and therapeutic domain coupled). In these assays, a positively charged substrate is phosphorylated with a radiolabeled phosphate group from an ATP if the kinase is not inhibited by an inhibitor peptide. The positively charged substrate is attracted to a negatively charged filter membrane, quantified with a scintillation counter, and compared to a 100% activity control. ATP concentrations within 15 μM of the apparent Km for ATP were chosen because an ATP concentration near the Km allowed for the kinases to have the same relative phosphorylation activity and because Hayess and Benndorf showed that the mechanism of the original inhibitor peptide was not to compete with the ATP binding site [40]. Individual conditions for each assay (reference inhibitors, buffer conditions, ATP concentration, substrate, etc.) and information about each kinase tested can be found on Millipore’s website at IC50 values for inhibitor peptides were determined using Millipore’s IC50Profiler Express service. The IC50 value was estimated from a 10-point curve of one-half log dilutions. For peptides that were tested for specificity, the concentration that inhibited approximately 95% of MK2 activity was chosen to profile against a battery of kinases related to MK2, cell viability, or human disease from Millipore’s KinaseProfiler service. In both assays, compounds were supplied in DMSO. Every kinase activity measurement was conducted in duplicate.

Mesothelial cell culture

Immortalized human pleural mesothelial cells (ATCC CRL-9444) were grown in Medium199 with Earle’s BSS and 0.75mM L-glutamine (Mediatech, Inc.), 1.25g/L sodium bicarbonate (Sigma), 3.3nM epidermal growth factor (EGF) (MBL International), 40 nM hydrocortisone (Sigma), 870nM insulin (MBL International), 20mM HEPES (Sigma), trace elements mixture B (Mediatech, Inc.), 10% fetal bovine serum (FBS) (Hyclone), 1% penicillin/streptomycin (Mediatech, Inc.). Passage number four mesothelial cells were used in live-dead assays.

Live-dead assay

Live-dead assays based on Molecular Probe’s LIVE/DEADR Viability/Cytotoxicity Kit for mammalian cells were used to assess the toxicity of various MK2 inhibitor peptides on human pleural mesothelial cells. Mesothelial cells were seeded in a Corning CellBindR, black well, clear-bottom, 96-well plate. Upon reaching 90–100% cell confluence, 200μL of the appropriate media with treatment was added to each well. Cells were incubated at 37°C and 5% CO2 for 24 hours. One-half hour prior to the 24-hour time point, 70% methanol was added to untreated cells to kill and permeabilize the cells. These cells were used for determining the background of Calcium-AM (CA) and the maximum dead signal for Ethidium homodimer-1 (EthD-1). Conversely, untreated live cells were used to determine the background of EthD-1 and the maximum live signal for Calcein-AM. At the 24-hour time interval, the cells were washed two times with 200μL of PBS. Then, 100μL of the appropriate stain stock was added to each well. Controls received the optimal concentration of either EthD-1 (8μM) or CA (4μM). All treated wells received the same optimal concentrations of EthD-1 and CA in the same stock. Samples were incubated for the appropriate optimal time interval at 37°C and 5% CO2. The optimal stain concentrations and time intervals were determined via staining confluent cells with variable concentrations of stain and measuring fluorescence for each stain at variable time intervals. Thus, the optimal time for dye incubation was chosen as the time that allowed for EthD-1 saturation and was still in the linear range of CA. All fluorescence was measured with a Spectramax M5 Microplate Reader (Molecular Devices). CA required an excitation wavelength of 494nm and an emission wavelength of 517nm. EthD-1 required an excitation wavelength of 528nm and an emission wavelength of 617nm.


Amino acid substitutions and deletions

Using a fluorescence-based kinase assay, peptides with select amino acid deletions and one alanine and D-amino acid substitution were examined (Table 1). The modifications to the original sequence described by Hayess and Benndorf were made for ease and cost of peptide synthesis. Both the D-amino acid and alanine substitutions showed that the asparagine was not critical for MK2 inhibition. In fact, replacing the asparagine with an alanine enhanced MK2 inhibition. Only moderate decreases in inhibition were seen when two N-terminal lysines were removed. Thus, further peptide studies were completed on peptides synthesized with only one N-terminal lysine and with either the asparagine from the original peptide or an alanine substituted for the asparagine.

Inhibition of MK2 – synergy between the CPP and therapeutic domains

The MK2 inhibitor peptide, KKKALNRQLGVAA, described by Hayess and Benndorf [40], was used as a control. The IC50 of the control peptide was compared to our novel cell permeant version containing the WLRRIKAWLRRI CPP. The cell permeant version dramatically enhanced MK2 inhibition when coupled with the therapeutic domain (Table 1). Since this CPP was not designed to inhibit MK2, we hypothesized that the CPP itself may serve as a general kinase inhibitor.

CPPs alone can inhibit MK2

To determine whether CPPs alone would inhibit MK2, we tested the ability of three CPPs to inhibit MK2 (Table 1 and Supplementary Figure 1). Of the three functional CPPs tested, YARAAARQARA [44] showed the least MK2 inhibition, and unlike the other CPPs tested, the level of MK2 inhibition did not vary over the concentration range investigated. One of the most widely known and used CPPs, YGRKKRRQRRR, inhibited 61.2% of MK2 activity at a concentration of 100 μM. Even at a concentration of 25 μM, this CPP inhibited 47.7% of MK2 activity. While minimal MK2 inhibition occurred with YARAAARQARA, the CPP WLRRIKAWLRRIKA potently inhibited MK2. In fact, WLRRIKAWLRRIKA was a much more potent inhibitor of MK2 activity than any therapeutic domain sequence. Interestingly, WLRRIKA, a truncated version of the CPP WLRRIKAWLRRI, was a very poor inhibitor of MK2. Overall, the functional CPPs tested had varying ability to inhibit MK2.

CPP choice affects potency

In five complete peptide inhibitors of MK2, CPP choice dramatically affected potency (Table 2). The IC50 of the original peptide developed by Hayess and Benndorf was 31 μM (IC50 curve not shown). Thus, all CPPs displayed synergistic efficacy with their respective therapeutic domains. However, the WLRRIKAWLRRI CPP had an IC50 value more than one order of magnitude lower than the peptide with the same therapeutic domain coupled to the YARAAARQARA CPP.

Table 2
Effect of CPP on IC50 of MK2 inhibitor peptides

CPP choice and substitution of asparagine for alanine affect specificity

The three most potent inhibitors of MK2 (Table 2) were tested against 43 diverse human kinases related to MK2, cell viability, or human disease at a single concentration that yielded 2–8% of normal MK2 activity (Table 3 and Supplementary Table 1). Even at over three times the concentration, peptides with the FAKLAARLYR and KAFAKLAARLYR CPPs were more specific than the peptide with the WLRRIKAWLRRI CPP.

Table 3
Effect of CPP on inhibitor peptide specificity for MK2

To further explore this specificity phenomenon, five complete inhibitor peptides were tested against 10 human kinases (Table 4). Based on the results of the radiometric and fluorometric assays, the YARAAARQARA CPP was a poor inhibitor of MK2 and, thus, may generally be a poor kinase inhibitor. Also, while the substitution of an alanine for an asparagine had only a modest effect on MK2 inhibition, we wanted to evaluate the effect of this substitution on specificity. The kinases chosen for this testing were selected for the following reasons. Three of the selected kinases are structurally similar to MK2 and, therefore, may also be inhibited by the MK2 inhibitor peptides; thus, data with different CPPs would indicate whether the CPP affected relative activity and specificity within kinase families. MAPKAP-K3 (MK3) shares 75% of MK2’s amino acid identity, and MK2 and MK3 phosphorylate many of the same substrates with similar kinetics [26]. MK2 and MK3 share 35–40% identity with CaMKI [49, 50]. MK5 shares 40% amino acid identity with MK2 and MK3 [19]. SAPK2a (equivalent to p38α MAP kinase) was selected to determine if the peptide inhibited the upstream kinase that phosphorylates MK2 in vivo [5154]. MLCK is a substrate of MK2 in vitro that was inhibited strongly in our previous kinase screening [55]. Thus, we were able to investigate the effects of the CPPs within a kinase signaling cascade. Finally, IRAK4, PKBβ, PKCδ, and ROCK-I were selected because they represent a diverse array of kinases and because the three peptides listed in Table 3 and tested previously dramatically inhibited their kinase activity.

Table 4
Effect of 5 complete inhibitor peptide variants on 10 human kinases

The results of this study demonstrated dramatic differences between the specificity of the five tested kinase inhibitor variants (Table 4). The concentrations selected for the assay yielded between 0–10% MK2 activity according to IC50 data (Table 2). The peptides in the first two peptide columns in Table 4 show a direct comparison of the inhibitors with the WLRRIKAWLRRI and YARAAARQARA CPPs and the arginine containing inhibitor peptide domain. For 8 of the 10 kinases, MK3 and CaMKI as the exceptions, the YARAAARQARA CPP containing inhibitor peptide showed significantly reduced nonspecific inhibition. Even with the more specific YARAAARQARA CPP, inhibition of MK3 and CaMKI is not surprising since these kinases are part of the same kinase family and have significant sequence homology as described above. The next three peptides in Table 4 provide a direct comparison of YARAAARQARA, FAKLAARLYR, and KAFAKLAARLYR CPPs with the inhibitor domain containing the asparagine to alanine substitution. Even with the asparagine to alanine substitution, the YARAAARQARA containing inhibitor shows increased specificity as the other two peptides antagonize MK5 activity and suppress PKCδ activity while YARAAARQARA containing peptides do not. Finally, comparison of the two YARAAARQARA containing peptides shows that while the asparagine to alanine amino acid substitution does not affect MK2 inhibition, it dramatically affects specificity. While this substitution affects specificity, the YARAAARQARA containing peptide even with the alanine substitution is significantly more specific than the WLRRIKAWLRRI-peptide containing the original asparagines residue. All peptides inhibited MK2 and MK3; however, in each case, the inhibition of MK2 was greater than that of MK3. Also, inhibition of CaMKI was equal to or greater than MK2 inhibition. None of the inhibitors except perhaps FAKLAARLYRKALARQLGVAA was a good inhibitor of SAPK2a (p38α).

In this study, CPPs that provided more specificity were used at higher concentrations to achieve the same level of inhibition of MK2. Even at three to ten times the concentrations of the other peptides, the peptides with the YARAAARQARA CPP inhibited IRAK4, MLCK, and PKCδ to a lesser degree. Also, although WLRRIKAWLRRIKALNRQLGVAA was used at the lowest concentration, this peptide had the least specificity.

Peptide specificity correlates with peptide toxicity

Table 5 shows that less specific peptides were much more lethal. The peptide with the WLRRIKAWLRRI CPP killed nearly every cell within 24hrs at a concentration of only 40μM. However, the CPP that caused the complete peptide to be most specific, YARAAARQARA, could be used at concentrations in excess of the highest peptide concentration tested, 3mM without resultant cell death.

Table 5
Effect of complete inhibitor peptide on mesothelial cell viability


Clearly, these kinase assays have limitations that preclude a direct correlation of the results with in vivo activity. The kinase profiling was performed on individual kinases at one concentration while cells contain a plethora of kinases with which the inhibitor may interact. Furthermore, kinase expression varies widely within and between cells. The described studies did not examine whether inhibition of MK2 would be preferred over inhibition of other kinases, thus, potentially affecting specificity. While these studies provided a great deal of useful information about kinase and peptide inhibitor interactions, more studies in competitive in vitro environments and in vivo will be needed to establish the therapeutic value of these peptide-based kinase inhibitors.

While this study focused primarily on the effects of the CPP on the specificity of potency of kinase-inhibitor peptides, even single amino acid substitutions within the kinase inhibition domain of the peptide can significantly impact specificity. Both fluorescence-based and radiometric assays demonstrated that an asparagine to alanine substitution in the therapeutic domain had minimal impact on inhibitor potency for MK2 (the minor activity differences seen between fluormetric and radiometric assay results can be explained by slight differences in assay conditions and ATP concentrations). However, the radiometric assay showed that this substitution decreased the specificity of the peptide. The drastic change in specificity caused by this modification would be nearly impossible to predict a priori.

The more surprising results came from examining CPPs used to deliver the therapeutic domain. CPPs alone inhibited varying amounts of MK2 activity. All assays also showed that the conjugation of each tested CPP to the MK2 inhibition sequence enhanced the efficacy of the therapeutic domain. Investigators have reported this phenomenon with other CPPs [56]. At this point, the mechanism of this enhanced efficacy is unclear. The CPP may help block the ATP binding site, enhance the binding of the therapeutic domain to the substrate binding site, bind to regulatory allosteric sites, or simply change the conformation of the kinase to reduce kinase activity. The crystal structure of MK2 reveals that the C-terminal regulatory domain of MK2 has high helical character, occupies the substrate binding pocket, and may act as a pseudosubstrate [57]. While the CPPs in this paper share little sequence homology with MK2’s C-terminal regulatory domain, they probably do form helix configurations and may enhance peptide binding to the substrate binding site of MK2 [44, 57]. The CPPs themselves do not appear to act as substrate mimics for MK2 since they share very little homology to known substrates of MK2 [58].

Perhaps, more importantly, this is the first report to our knowledge showing that CPP selection can dramatically affect peptide specificity in addition to activity. Moreover, while these peptides inhibited additional kinases within the MK2 family, the peptides also inhibited kinases with diverse structures and evolutionary origin. While these assays showed that no peptide was completely specific for MK2, even small molecular inhibitors of kinase activity approved by the United States FDA lack specificity in similar kinase activity assays [5962]. Complete specificity for a target kinase may not even be desirable since inhibiting a single kinase may not be sufficient to counter the actions of compensatory pathways.

The data presented cannot explain why different CPPs lead to varying specificity and IC50s, but both charge and hydrophobicity likely contribute to this phenomenon. While the mechanism of entry is believed to vary for different CPPs, most CPPs are positively charged and have electrostatic interactions with the phospholipids, gangliosides, glycosaminoglycans, and polysialic acid attached to the plasma membrane [6366]. In addition to charge, hydrophobicity has been shown to be important for cell penetration [67]. Thus, the identity of hydrophobic amino acid within the primary sequence of the CPPs may also play a role in therapeutic potency and specificity. Nonetheless, we have shown not only that the sequence of the inhibitor domain affects kinase specificity, but also that the choice of CPP coveys dramatically different pharmacological activity to kinase inhibitor peptides. Future studies will examine the role of charged and hydrophobic residues within the primary structure of CPPs to further elucidate the phenomenon that leads to change in therapeutic potency and toxicity of the delivered therapeutic.

Of the peptides tested, less specific peptides were much more lethal to pleural mesothelial cells. The reason that more specific peptides could be used at higher concentrations is not clear. One would expect peptides with higher specificity to inhibit fewer kinases crucial to cell function inducing less toxicity. The fact that knocking out MK2 is not lethal in mice indicates that even extremely high concentrations of a specific inhibitor of MK2 should not be lethal and adds support to this theory [29, 68]. In contrast, investigators generally agree that different peptide CPPs enter cells in variable ways [14, 15, 69]. Thus, the lethality of less specific peptides may not be only a function of lack of inhibitor specificity but also a function of the peptide’s ability to enter the cell cytoplasm. Finally, both arginine and tyrosine contribute to nonspecific protein-protein interactions, and this phenomenon may also be playing a role in both specificity and toxicity of the peptides. Regardless, CPP choice was largely responsible for increased inhibitor peptide toxicity. Peptides with identical therapeutic domains but different CPPs had vastly different toxicity profiles.

We believe that these results have wider implications for delivering cargos with CPPs. The CPPs themselves may inhibit multiple kinases in the cell. One of the most widely known and used CPPs, YGRKKRRQRRR, had a substantial impact on MK2 activity. Thus, the results of cell and animal studies using CPPs have likely been somewhat confounded by the biological activity of the CPP in cells. Furthermore, these studies show that CPP selection can have a dramatic impact on therapeutic specificity and can actually be used to enhance the efficacy of therapeutic cargos. Additionally, less specific peptides that differed from more specific peptides only in CPP identity were lethal to cells at lower concentrations versus more specific peptides. As a result, future work will warrant careful consideration of the diverse properties of CPPs as well as judicious selection of a CPP for specific applications.

Supplementary Material

Supp Fig 1

Supp Info



This research was supported in part by the NIH (National Institutes of Health; Bethesda, MD, U.S.A.) (grant number K25HL074968) and the Purdue Research Foundation Trask Innovation Fund. B.W. was supported in part by an NIH Medical Scientist Training Program grant (grant number GM077229) and by the Purdue Research Foundation.


cell penetrating peptide
transforming growth factor beta-1
tumor necrosis factor-alpha
granulocyte macrophage-colony stimulating factor
Food and Drug Administration
Matrix-assisted laser desorption/ionization time of flight
mitogen-activated protein kinase-activated protein kinase 2 (also commonly known as MAPKAP-K2
mitogen-activated protein kinase-activated protein kinase 3
Calcium/calmodulin-dependent protein kinase I
mitogen-activated protein kinase-activated protein kinase 5 (also commonly known as PRAK)
mitogen-activated protein kinase 14 subunit alpha (also commonly known as p38α or MAPK14α)
mitogen-activated protein kinase 14 subunit beta (also commonly known as SAPK2b or MAPK14β)
interleukin-1 receptor-associated kinase 4
myosin light chain kinase (also commonly known as MYLK)
thymoma viral proto-oncogene 2 (also commonly known as Akt2)
Protein kinase C delta
Rho-associated coiled-coil containing protein kinase 1



A. Panitch, B.L. Seal, and C.M. Brophy have a financial conflict of interest. Moerae Matrix, Inc has an option to license the technology described in the manuscript, and A. Panitch, B.L. Seal, and C.M. Brophy own greater than 5% interest in the company.


1. Flynn CR, Komalavilas P, Tessier D, Thresher J, Niederkofler EE, Dreiza CM, Nelson RW, Panitch A, Joshi L, Brophy CM. Transduction of biologically active motifs of the small heat shock-related protein, HSP20, leads to relaxation of vascular smooth muscle. Faseb Journal. 2003;17 [PubMed]
2. Dreiza CM, Brophy CM, Komalavilas P, Furnish EJ, Joshi L, Pallero MA, Murphy-Ullrich JE, von Rechenberg M, Ho YS, Richardson B, Xu N, Zhen Y, Peltier JM, Panitch A. Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics. Faseb J. 2005;19:261–263. [PubMed]
3. Lopes LB, Flynn C, Komalavilas P, Panitch A, Brophy CM, Seal BL. Inhibition of HSP27 phosphorylation by a cell-permeant MAPKAP Kinase 2 inhibitor. Biochem Biophys Res Commun. 2009;382:535–539. [PMC free article] [PubMed]
4. Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell. 1988;55:1189–1193. [PubMed]
5. Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell. 1988;55:1179–1188. [PubMed]
6. Saalik P, Elmquist A, Hansen M, Padari K, Saar K, Viht K, Langel U, Pooga M. Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjug Chem. 2004;15:1246–1253. [PubMed]
7. Tkachenko AG, Xie H, Liu Y, Coleman D, Ryan J, Glomm WR, Shipton MK, Franzen S, Feldheim DL. Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains. Bioconjug Chem. 2004;15:482–490. [PubMed]
8. Leifert JA, Harkins S, Whitton JL. Full-length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity. Gene Ther. 2002;9:1422–1428. [PubMed]
9. Violini S, Sharma V, Prior JL, Dyszlewski M, Piwnica-Worms D. Evidence for a plasma membrane-mediated permeability barrier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate. Biochemistry. 2002;41:12652–12661. [PubMed]
10. Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF. Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Research. 2001;61:474–477. [PubMed]
11. Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci U S A. 2000;97:13003–13008. [PubMed]
12. Derossi D, Joliot AH, Chassaing G, Prochiantz A. The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem. 1994;269:10444–10450. [PubMed]
13. Joliot A, Pernelle C, Deagostini-Bazin H, Prochiantz A. Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci U S A. 1991;88:1864–1868. [PubMed]
14. Joliot A, Prochiantz A. Transduction peptides: from technology to physiology. Nat Cell Biol. 2004;6:189–196. [PubMed]
15. Gump JM, Dowdy SF. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 2007;13:443–448. [PubMed]
16. El-Andaloussi S, Holm T, Langel U. Cell-penetrating peptides: mechanisms and applications. Curr Pharm Des. 2005;11:3597–3611. [PubMed]
17. Gaestel M. MAPKAP kinases - MKs - two’s company, three’s a crowd. Nat Rev Mol Cell Biol. 2006;7:120–130. [PubMed]
18. Ono K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal. 2000;12:1–13. [PubMed]
19. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev. 2004;68:320–344. [PMC free article] [PubMed]
20. Kayyali US, Pennella CM, Trujillo C, Villa O, Gaestel M, Hassoun PM. Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2. J Biol Chem. 2002;277:42596–42602. [PubMed]
21. Tilly BC, Gaestel M, Engel K, Edixhoven MJ, de Jonge HR. Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade. FEBS Lett. 1996;395:133–136. [PubMed]
22. Engel K, Ahlers A, Brach MA, Herrmann F, Gaestel M. MAPKAP kinase 2 is activated by heat shock and TNF-alpha: in vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade. J Cell Biochem. 1995;57:321–330. [PubMed]
23. Belka C, Ahlers A, Sott C, Gaestel M, Herrmann F, Brach MA. Interleukin (IL)-6 signaling leads to phosphorylation of the small heat shock protein (Hsp)27 through activation of the MAP kinase and MAPKAP kinase 2 pathway in monocytes and monocytic leukemia cells. Leukemia. 1995;9:288–294. [PubMed]
24. Ahlers A, Engel K, Sott C, Gaestel M, Herrmann F, Brach MA. Interleukin-3 and granulocyte-macrophage colony-stimulating factor induce activation of the MAPKAP kinase 2 resulting in in vitro serine phosphorylation of the small heat shock protein (Hsp 27) Blood. 1994;83:1791–1798. [PubMed]
25. Ahlers A, Belka C, Gaestel M, Lamping N, Sott C, Herrmann F, Brach MA. Interleukin-1-induced intracellular signaling pathways converge in the activation of mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 and the subsequent phosphorylation of the 27-kilodalton heat shock protein in monocytic cells. Mol Pharmacol. 1994;46:1077–1083. [PubMed]
26. Clifton AD, Young PR, Cohen P. A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress. FEBS Lett. 1996;392:209–214. [PubMed]
27. Kotlyarov A, Gaestel M. Is MK2 (mitogen-activated protein kinase-activated protein kinase 2) the key for understanding post-transcriptional regulation of gene expression? Biochem Soc Trans. 2002;30:959–963. [PubMed]
28. Ronkina N, Kotlyarov A, Gaestel M. MK2 and MK3--a pair of isoenzymes? Front Biosci. 2008;13:5511–5521. [PubMed]
29. Kotlyarov A, Neininger A, Schubert C, Eckert R, Birchmeier C, Volk HD, Gaestel M. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol. 1999;1:94–97. [PubMed]
30. Tietz AB, Malo A, Diebold J, Kotlyarov A, Herbst A, Kolligs FT, Brandt-Nedelev B, Halangk W, Gaestel M, Goke B, Schafer C. Gene deletion of MK2 inhibits TNF-alpha and IL-6 and protects against cerulein-induced pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2006;290:G1298–1306. [PubMed]
31. Gorska MM, Liang Q, Stafford SJ, Goplen N, Dharajiya N, Guo L, Sur S, Gaestel M, Alam R. MK2 controls the level of negative feedback in the NF-kappaB pathway and is essential for vascular permeability and airway inflammation. J Exp Med. 2007;204:1637–1652. [PMC free article] [PubMed]
32. Jagavelu K, Tietge UJ, Gaestel M, Drexler H, Schieffer B, Bavendiek U. Systemic deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in hypercholesterolemic mice. Circ Res. 2007;101:1104–1112. [PubMed]
33. Culbert AA, Skaper SD, Howlett DR, Evans NA, Facci L, Soden PE, Seymour ZM, Guillot F, Gaestel M, Richardson JC. MAPK-activated protein kinase 2 deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity. Relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease. J Biol Chem. 2006;281:23658–23667. [PubMed]
34. Thomas T, Hitti E, Kotlyarov A, Potschka H, Gaestel M. MAP-kinase-activated protein kinase 2 expression and activity is induced after neuronal depolarization. Eur J Neurosci. 2008;28:642–654. [PubMed]
35. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell. 2007;11:175–189. [PMC free article] [PubMed]
36. Hegen M, Gaestel M, Nickerson-Nutter CL, Lin LL, Telliez JB. MAPKAP kinase 2-deficient mice are resistant to collagen-induced arthritis. J Immunol. 2006;177:1913–1917. [PubMed]
37. Kontoyiannis D, Boulougouris G, Manoloukos M, Armaka M, Apostolaki M, Pizarro T, Kotlyarov A, Forster I, Flavell R, Gaestel M, Tsichlis P, Cominelli F, Kollias G. Genetic dissection of the cellular pathways and signaling mechanisms in modeled tumor necrosis factor-induced Crohn’s-like inflammatory bowel disease. J Exp Med. 2002;196:1563–1574. [PMC free article] [PubMed]
38. Gaestel M, Mengel A, Bothe U, Asadullah K. Protein kinases as small molecule inhibitor targets in inflammation. Curr Med Chem. 2007;14:2214–2234. [PubMed]
39. Lopes LB, Flynn C, Komalavilas P, Panitch A, Brophy CM, Seal BL. Inhibition of HSP27 phosphorylation by a cell-permeant MAPKAP Kinase 2 inhibitor. Biochemical and Biophysical Research Communications. In Press, Uncorrected Proof. [PMC free article] [PubMed]
40. Hayess K, Benndorf R. Effect of protein kinase inhibitors on activity of mammalian small heat-shock protein (HSP25) kinase. Biochem Pharmacol. 1997;53:1239–1247. [PubMed]
41. Lukas TJ, Mirzoeva S, Slomczynska U, Watterson DM. Identification of novel classes of protein kinase inhibitors using combinatorial peptide chemistry based on functional genomics knowledge. J Med Chem. 1999;42:910–919. [PubMed]
42. Schwarze SR, Hruska KA, Dowdy SF. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 2000;10:290–295. [PubMed]
43. Becker-Hapak M, McAllister SS, Dowdy SF. TAT-mediated protein transduction into mammalian cells. Methods. 2001;24:247–256. [PubMed]
44. Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF. Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res. 2001;61:474–477. [PubMed]
45. Wadia JS, Dowdy SF. Protein transduction technology. Curr Opin Biotechnol. 2002;13:52–56. [PubMed]
46. Seal BL, Panitch A. Physical polymer matrices based on affinity interactions between peptides and polysaccharides. Biomacromolecules. 2003;4:1572–1582. [PubMed]
47. Tyler-Cross R, Sobel M, McAdory LE, Harris RB. Structure-function relations of antithrombin III-heparin interactions as assessed by biophysical and biological assays and molecular modeling of peptide-pentasaccharide-docked complexes. Arch Biochem Biophys. 1996;334:206–213. [PubMed]
48. Bodanszky M. Peptide chemistry: a practical textbook. Springer-Verlag; Berlin, New York: 1988.
49. Zu YL, Wu F, Gilchrist A, Ai Y, Labadia ME, Huang CK. The primary structure of a human MAP kinase activated protein kinase 2. Biochem Biophys Res Commun. 1994;200:1118–1124. [PubMed]
50. Stokoe D, Caudwell B, Cohen PT, Cohen P. The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2. Biochem J. 1993;296(Pt 3):843–849. [PubMed]
51. Frodin M, Antal TL, Dummler BA, Jensen CJ, Deak M, Gammeltoft S, Biondi RM. A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. Embo J. 2002;21:5396–5407. [PubMed]
52. Guay J, Lambert H, Gingras-Breton G, Lavoie JN, Huot J, Landry J. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci. 1997;110(Pt 3):357–368. [PubMed]
53. McLaughlin MM, Kumar S, McDonnell PC, Van Horn S, Lee JC, Livi GP, Young PR. Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J Biol Chem. 1996;271:8488–8492. [PubMed]
54. Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, Nebreda AR. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell. 1994;78:1027–1037. [PubMed]
55. Haydon CE, Watt PW, Morrice N, Knebel A, Gaestel M, Cohen P. Identification of a phosphorylation site on skeletal muscle myosin light chain kinase that becomes phosphorylated during muscle contraction. Arch Biochem Biophys. 2002;397:224–231. [PubMed]
56. Dostmann WR, Taylor MS, Nickl CK, Brayden JE, Frank R, Tegge WJ. Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Ialpha inhibit NO-induced cerebral dilation. Proc Natl Acad Sci U S A. 2000;97:14772–14777. [PubMed]
57. Meng W, Swenson LL, Fitzgibbon MJ, Hayakawa K, Ter Haar E, Behrens AE, Fulghum JR, Lippke JA. Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export. J Biol Chem. 2002;277:37401–37405. [PubMed]
58. Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AE, Yaffe MB. MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Mol Cell. 2005;17:37–48. [PubMed]
59. Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT, Faraoni R, Floyd M, Hunt JP, Lockhart DJ, Milanov ZV, Morrison MJ, Pallares G, Patel HK, Pritchard S, Wodicka LM, Zarrinkar PP. A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol. 2008;26:127–132. [PubMed]
60. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000;351:95–105. [PubMed]
61. Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an update. Biochem J. 2003;371:199–204. [PubMed]
62. Fabian MA, Biggs WH, 3rd, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, Carter TA, Ciceri P, Edeen PT, Floyd M, Ford JM, Galvin M, Gerlach JL, Grotzfeld RM, Herrgard S, Insko DE, Insko MA, Lai AG, Lelias JM, Mehta SA, Milanov ZV, Velasco AM, Wodicka LM, Patel HK, Zarrinkar PP, Lockhart DJ. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol. 2005;23:329–336. [PubMed]
63. Derossi D, Chassaing G, Prochiantz A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 1998;8:84–87. [PubMed]
64. Rothbard JB, Kreider E, VanDeusen CL, Wright L, Wylie BL, Wender PA. Arginine-rich molecular transporters for drug delivery: role of backbone spacing in cellular uptake. J Med Chem. 2002;45:3612–3618. [PubMed]
65. Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB. Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res. 2000;56:318–325. [PubMed]
66. Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, Sugiura Y. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem. 2001;276:5836–5840. [PubMed]
67. Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. The Journal of biological chemistry. 1995;270:14255–14258. [PubMed]
68. Lehner MD, Schwoebel F, Kotlyarov A, Leist M, Gaestel M, Hartung T. Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection. J Immunol. 2002;168:4667–4673. [PubMed]
69. Prochiantz A. For protein transduction, chemistry can win over biology. Nat Methods. 2007;4:119–120. [PubMed]