Utilization of a cell-penetrating peptide-adaptor for delivery of human papillomavirus protein E2 into cervical cancer cells to arrest cell growth and promote cell death.

BACKGROUND: Human papillomavirus (HPV) is the causative agent of nearly all forms of cervical cancer, which can arise upon viral integration into the host genome and concurrent loss of viral regulatory gene E2. Gene-based delivery approaches show that E2 reintroduction reduces proliferative capacity and promotes apoptosis in vitro. AIMS: This work explored if our calcium-dependent protein-based delivery system, TAT-CaM, could deliver functional E2 protein directly into cervical cancer cells to limit proliferative capacity and induce cell death. MATERIALS AND RESULTS: TAT-CaM and the HPV16 E2 protein containing a CaM-binding sequence (CBS-E2) were expressed and purified from Escherichia coli. Calcium-dependent binding kinetics were verified by biolayer interferometry. Equimolar TAT-CaM:CBS-E2 constructs were delivered into the HPV16+ SiHa cell line and uptake verified by confocal microscopy. Proliferative capacity was measured by MTS assay and cell death was measured by release of lactate dehydrogenase. As a control, human microvascular cells (HMECs) were used. As expected, TAT-CaM bound CBS-E2 with high affinity in the presence of calcium and rapidly disassociated upon its removal. After introduction by TAT-CaM, fluorescently labeled CBS-E2 was detected in cellular interiors by orthogonal projections taken at the depth of the nucleus. In dividing cells, E2 relocalized to regions associated with the mitotic spindle. Cells receiving a daily dose of CBS-E2 for 4 days showed a significant reduction in metabolic activity at low doses and increased cell death at high doses compared to controls. This phenotype was retained for 7 days with no further treatments. When subcultured on day 12, treated cells regained their proliferative capacity. CONCLUSIONS: Using the TAT-CaM platform, bioactive E2 protein was delivered into living cervical cancer cells, inducing senescence and cell death in a time- and dose-dependent manner. These results suggest that this nucleic acid and virus-free delivery method could be harnessed to develop novel, effective protein therapeutics.

MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites.

Nitric oxide synthase 3 (NOS3) is a major vasoprotective enzyme that catalyzes the conversion of l-arginine to nitric oxide (NO) in response to a significant number of signaling pathways. Here, we provide evidence that NOS3 interactions with MAP kinases have physiological relevance. Binding interactions of NOS3 with c-Jun N-terminal kinase (JNK1α1 ), p38α, and ERK2 were characterized using optical biosensing with full-length NOS3 and NOS3 specific peptides and phosphopeptides. Like p38α and ERK2, JNK1α1 exhibited high-affinity binding to full-length NOS3 (KD 15 nm). Rate constants exhibited fast-on, slow-off binding (kon = 4106 m-1 s-1 ; koff = 6.2 × 10-5 s-1 ). Further analysis using synthetic NOS3 peptides revealed two MAP kinase binding sites unique to NOS3. p38α evinced similar affinity with both NOS3 binding sites. For ERK2 and JNK1α1, the affinity at the two sites differed. However, NOS3 peptides with a phosphate at either S114 or S633 did not meaningfully interact with the kinases. Immunoblotting revealed that each kinase phosphorylated NOS3 with a unique pattern. JNK1α1 predominantly phosphorylated NOS3 at S114, ERK2 at S600, and p38α phosphorylated both residues. In vitro production of NO was unchanged by phosphorylation at these sites. In human microvascular endothelial cells, endogenous interactions of all the MAP kinases with NOS3 were captured using proximity ligation assay in resting cells. Our results underscore the importance of MAP kinase interactions, identifying two unique NOS3 interaction sites with potential for modulation by MAP kinase phosphorylation (S114) and other signaling inputs, like protein kinase A (S633).

Detection of Nitric Oxide by Membrane Inlet Mass Spectrometry.

Membrane inlet mass spectrometry (MIMS) is a reproducible and reliable method for the measurement of nitric oxide in aqueous solution with a lower limit of detection of 10 nM and a linear response to 50 µM. MIMS utilizes a semipermeable membrane to partition analytes based on physicochemical properties from the bulk sample into the mass spectrometer. Silastic tubing allows the introduction of small gaseous molecules including nitric oxide (NO) into the high vacuum of a mass spectrometer. We describe the measurement of NO generated chemically from nitrite and MAHMA NONOate as well as enzymatically by nitric oxide synthase (NOS).

A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations.

Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this 'endosomal escape problem.' Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells.

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos.

The use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602.

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2-3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.

MAP kinases bind endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) contains a motif similar to recognition sequences in known MAPK binding partners. In optical biosensing experiments, eNOS bound p38 and ERK with ∼100 nM affinity and complex kinetics. Binding is diffusion-limited (k on âˆ¼ .15 × 10(6) M(-1) s(-1)). Neuronal NOS also bound p38 but exhibited much slower and weaker binding. p38-eNOS binding was inhibited by calmodulin. Evidence for a ternary complex was found when eNOS bound p38 was exposed to CaM, increasing the apparent dissociation rate. These observations strongly suggest a direct role for MAPK in regulation of NOS with implications for signaling pathways including angiogenesis and control of vascular tone.

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin.

Using interferometry-based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smoluchowski limit imposed by orientation-independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM-binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495, adjacent to the CaM-binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca(2+) binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca(2+) dependence of CaM binding. Hence, changes in the Ca(2+) sensitivity of CaM binding always imply changes in the NOS-CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca(2+) sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.

Inhibition of tristetraprolin deadenylation by poly(A) binding protein.

Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF.

MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages.

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.

Loss of MNK function sensitizes fibroblasts to serum-withdrawal induced apoptosis.

Map kinase-interacting protein kinases 1 and 2 (MNK1, MNK2) function downstream of p38 and ERK MAP kinases, but there are large gaps in our knowledge of how MNKs are regulated and function. Mice deleted of both genes are apparently normal, suggesting that MNKs function in adaptive pathways during stress. Here, we show that mouse embryo fibroblasts (MEFs) obtained from mnk1 (-/-)/mnk2 (-/-) as well as mnk1 (-/-) and mnk2 (-/-) mice are sensitized to caspase-3 activation upon withdrawal of serum in comparison to wild-type cells. Caspase-3 cleavage occurs with all cells in the panel, but most rapidly and robustly in cells derived from mice lacking both MNK genes. Treatment of wild-type MEFs in the panel with a compound (CGP57380) that inhibits MNK1 and MNK2 sensitizes wild-type cells for serum-withdrawal induced apoptosis, suggesting that sensitization is due to loss of MNK function and not to a secondary event. Reintroduction of wild-type MNK1 in the double knockout MEFs results in decreased sensitivity to serum withdrawal that is not observed for wild-type MNK2, or the kinase dead variant. Our work identifies MNKs as kinases involved in anti-apoptotic signaling in response to serum withdrawal.

MNK1 and MNK2 regulation in HER2-overexpressing breast cancer lines.

MAPK-interacting protein kinases 1 and 2 (MNK1 and MNK2) function downstream of p38 and ERK MAPK, but there are large gaps in our knowledge of how MNKs are regulated and function. As proteins activated in the HER2/Ras/Raf/ERK pathway, the MNKs are of potential interest in HER2-overexpressing cancers. We utilized a panel of breast cell lines (HCC1419, AU565, SKBR3, MCF7, and MCF10A), three of which overexpress HER2, to characterize the amounts and activation status of MNKs and other pathway enzymes (ERKs and RSKs) in these cells. We generated a phosphospecific antibody to Thr(P)-214 in the T-loop of MNKs and found that phosphorylations of both Thr-209 and Thr-214 in human MNK1 are required for activation. Increased phosphorylation and activity of the MNKs correlate with HER2 overexpression, and inhibition of the MNKs reduces colony formation in soft agar. Our work identifies the MNKs as potential therapeutic targets for breast cancer treatments.

The MAP kinase ERK5 binds to and phosphorylates p90 RSK.

We showed previously that p90 RSK was activated in cells expressing an activated mutant of MEK5, the activator of the MAP kinase ERK5. Based on the following evidence, we suggest that ERK5 can directly activate RSK in cells. ERK5 binds to RSK in vitro and co-immunoprecipitates from cell extracts; activation of ERK5 weakens its binding to RSK, suggesting that RSK is released upon activation. Phosphorylation of RSK by ERK5 in vitro causes its activation, indicating that RSK is a substrate of ERK5. In cells activation of ERK5 but not p38 or the c-Jun N-terminal kinase is associated with RSK activation. The large C-terminal domain of ERK5 is not required for binding or activation of RSK by ERK5; however, the common docking or CD domain of ERK5 and the docking or D domain of RSK are important for their association.

Glucose regulates interleukin-8 production in aortic endothelial cells through activation of the p38 mitogen-activated protein kinase pathway in diabetes.

We have shown that chronic elevated glucose (25 mm) increases monocyte adhesion to human aortic endothelial cells (EC). This increased adhesion is mediated primarily through induction of interleukin (IL)-8 via activation of the transcription factor AP-1 (Srinivasan, S., Yeh, M., Danziger, E. C., Hatley, M. E., Riggan, A. E., Leitinger, N., Berliner, J. A., and Hedrick, C. C. (2003) Circ. Res. 92, 371-377). In the current study, we identified the elements in the AP-1 transcriptional complex that are activated by glucose. These elements include c-Jun, c-Fos, and Fra-1. AP-1 is activated by cellular oxidative stress, and we have reported significant production of ROS by high glucose-cultured cells. We examined signaling pathways upstream of AP-1 in EC that lead to AP-1 activation by HG. EC cultured in 25 mm glucose had a 2-fold increase in p38 phosphorylation compared with control normal glucose-cultured EC. Inhibition of the p38 pathway using 5 microm SB203580 significantly reduced glucose-mediated IL-8 mRNA production by 60%. Furthermore, blocking p38 pathway activation using a dominant-negative p38 construct significantly reduced glucose-mediated monocyte adhesion by 50%. Thus, glucose-stimulated monocyte adhesion is primarily regulated through phosphorylation of p38 with subsequent activation of AP-1, leading to IL-8 production. To study this pathway in the setting of diabetes, we used the db/db mouse. P38 phosphorylation was increased in diabetic db/db mice compared with control mice. We found a dramatic elevation in plasma levels of KC, the mouse ortholog of IL-8 in diabetic db/db mice (1800 +/- 100 pg/ml KC in db/db versus 300 +/- 75 pg/ml in C57BL/6J control mice, p < 0.0001). Inhibition of the p38 pathway in diabetic db/db mice significantly reduced monocyte adhesion by 50%. Taken together, these data indicate that chronic elevated glucose in diabetes activates the p38 MAP kinase pathway to increase inflammatory IL-8 gene induction and monocyte/endothelial adhesion.

MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding.

MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3'-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc linked to magnetic beads). MK2Delta3B, activated in Escherichia coli by p38alpha, phosphorylates TTP in vitro at major sites Ser(52) and Ser(178) (>10-fold in abundance) as well as at several minor sites that were detected after enriching for phosphopeptides with immobilized metal affinity chromatography. MK2 phosphorylation of TTP creates a functional 14-3-3 binding site. In cells, TTP was phosphorylated at Ser(52), Ser(178), Thr(250), and Ser(316) and at SP sites in a cluster (Ser(80)/Ser(82)/Ser(85)). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of Ser(52) and Ser(178). Overexpression of MK2 sufficed to increase phosphorylation of Ser(52) and Ser(178) but not Ser(80)/Ser(82)/Ser(85) or Thr(250). Thus, Ser(52) and Ser(178) are putative MK2 sites in vivo. Identified phosphosite(s) may be biologic switches controlling mRNA stability and translation.

Characterization of the p90 ribosomal S6 kinase 2 carboxyl-terminal domain as a protein kinase.

The carboxyl-terminal domain (CTD) of the p90 ribosomal S6 kinases (RSKs) is an important regulatory domain in RSK and a model for kinase regulation of FXXFXF(Y) motifs in AGC kinases. Its properties had not been studied. We reconstituted activation of the CTD in Escherichia coli by co-expression with active ERK2 mitogen-activated protein kinase (MAPK). GST-RSK2-(aa373-740) was phosphorylated in the P-loop (Thr(577)) by MAPK, accompanied by increased phosphorylation on the hydrophobic motif site, Ser(386). Activated GST-RSK2-(aa373-740) phosphorylates synthetic peptides based on Ser(386). The peptide RRQLFRGFSFVAK, which was termed CTDtide, was phosphorylated with K(m) and V(max) values of approximately 140 microm and approximately 1 micromol/min/mg, respectively. Residues Leu at p -5 and Arg at p -3 are important for substrate recognition, but a hydrophobic residue at p +4 is not. RSK2 CTD is a much more selective peptide kinase than MAPK-activated protein kinase 2. CTDtide was used to probe regulation of hemagglutinin-tagged RSK proteins immunopurified from epidermal growth factor-stimulated BHK-21 cells. K100A but not K451A RSK2 phosphorylates CTDtide, indicating a requirement for the CTD. RSK2-(aa1-389) phosphorylates the S6 peptide, and this activity is inactivated by S386A mutation, but RSK2-(aa1-389) does not phosphorylate CTDtide. In contrast, RSK2-(aa373-740) containing only the CTD phosphorylates CTDtide robustly. Thus, CTDtide is phosphorylated by the CTD but not the NH(2)-terminal domain (NTD). Epidermal growth factor activates the CTD and NTD in parallel. Activity of the CTD for peptide phosphorylation correlates with Thr(577) phosphorylation. CTDtide activity is constrained in full-length RSK2. Interestingly, mutation of the conserved lysine in the ATP-binding site of the NTD completely eliminates S6 kinase activity, but a similar mutation of the CTD does not completely ablate kinase activity for intramolecular phosphorylation of Ser(386), even though it greatly reduces CTDtide activity. The standard lysine mutation used routinely to study kinase functions in vivo may be unsatisfactory when the substrate is intramolecular or in a tight complex.