PTPIP51 crosslinks the NFκB signaling and the MAPK pathway in SKBR3 cells.

AIM: PTPIP51 interacts with NFκB signaling at the RelA and IκB level. NFκB signaling is linked to the initiation, progression and metastasis of breast cancer. Her2-amplified breast cancer cells frequently display activation of the NFκB signaling. We aimed to clarify the effects of NFκB inhibition on the NFκB- and MAPK-related interactome of PTPIP51 and cell viability in HaCat cells and SKBR3 cells. RESULTS: IKK-16 selectively reduced cell viability in SKBR3 cells. PDTC induced a formation of the Raf1/14-3-3/PTPIP51 complex in SKBR3 cells, indicating a shift of PTPIP51 into MAPK signaling. CONCLUSION: IKK-16 selectively inhibits cell viability of SKBR3 cells. In addition, PTPIP51 might serve as the mediator between NFκB signaling and the MAPK pathway in SKBR3.

Crosstalks of the PTPIP51 interactome revealed in Her2 amplified breast cancer cells by the novel small molecule LDC3/Dynarrestin.

LDC3/Dynarrestin, an aminothiazole derivative, is a recently developed small molecule, which binds protein tyrosine phosphatase interacting protein 51 (PTPIP51). PTPIP51 interacts with various proteins regulating different signaling pathways leading to proliferation and migration. Her2 positive breast cancer cells (SKBR3) express high levels of PTPIP51. Therefore, we investigated the effects of LDC3/Dynarrestin on PTPIP51 and its interactome with 12 different proteins of various signal pathways including the interaction with dynein in SKBR3 cells. The localization and semi-quantification of PTPIP51 protein and the Tyr176 phosphorylated PTPIP51 protein were evaluated. Protein-protein-interactions were assessed by Duolink proximity ligation assays. Interactions and the activation of signal transduction hubs were examined with immunoblots. LDC3/Dynarrestin led to an increased PTPIP51 tyrosine 176 phosphorylation status while the overall amount of PTPIP51 remained unaffected. These findings are paralleled by an enhanced interaction of PTPIP51 with its crucial kinase c-Src and a reduced interaction with the counteracting phosphatase PTP1B. Furthermore, the treatment results in a significantly augmented interaction of PTPIP51/14-3-3ß and PTPIP51/Raf1, the link to the MAPK pathway. Under the influence of LDC3/Dynarrestin, the activity of the MAPK pathway rose in a concentration-dependent manner as indicated by RTK assays and immunoblots. The novel small molecule stabilizes the RelA/IκB/PTPIP51 interactome and can abolish the effects caused by TNFα stimulation. Moreover, LDC3/Dynarrestin completely blocked the Akt signaling, which is essential for tumor growth. The data were compared to the recently described interactome of PTPIP51 in LDC3/Dynarrestin treated non-cancerous keratinocyte cells (HaCaT). Differences were identified exclusively for the mitochondrial-associated ER-membranes (MAM) interactions and phospho-regulation related interactome of PTPIP51.LDC3/Dynarrestin gives the opportunity/possibility to influence the MAPK signaling, NFkB signaling and probably calcium homeostasis in breast cancer cells by affecting the PTPIP51 interactome.

Effectiveness of EGFR/HER2-targeted drugs is influenced by the downstream interaction shifts of PTPIP51 in HER2-amplified breast cancer cells.

Breast cancer is the most common female cancerous disease and the second most cause of cancer death in women. About 20-30% of these tumors exhibit an amplification of the HER2/ErbB2 receptor, which is coupled to a more aggressive and invasive growth of the cancer cells. Recently developed tyrosine kinase inhibitors and therapeutic antibodies targeting the HER2 receptor improved the overall survival time compared with sole radio- and chemotherapy. Upcoming resistances against the HER2-targeted therapy make a better understanding of the receptor associated downstream pathways an absolute need. In earlier studies, we showed the involvement of Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) in the mitogen-activated protein kinase (MAPK) pathway. The MAPK pathway is one of the most frequently overactivated pathways in HER2-amplified breast cancer cells. This study is aimed to elucidate the effects of four different TKIs on the interactome of PTPIP51, namely with the receptors EGFR and HER2, 14-3-3/Raf1 (MAPK pathway), its regulating enzymes, and the mitochondria-associated interaction partners in HER2 breast cancer cell lines (SK-BR3 and BT474) by using the Duolink proximity ligation assay, immunoblotting and knockdown of PTPIP51. Inhibition of both EGFR and HER2/ErbB2R shifted PTPIP51 into the MAPK pathway, but left the mitochondria-associated interactome of PTPIP51 unattended. Exclusively inhibiting HER2/ErbB2 by Mubritinib did not affect the interaction of PTPIP51 with the MAPK signaling. Selective inhibition of HER2 induced great alterations of mitochondria-associated interactions of PTPIP51, which ultimately led to the most-effective reduction of cell viability of SK-BR3 cells of all tested TKIs. The results clearly reveal the importance of knowing the exact mechanisms of the inhibitors affecting receptor tyrosine kinases in order to develop more efficient anti-HER2-targeted therapies.

PTPIP51­A New RelA-tionship with the NFκB Signaling Pathway.

The present study shows a new connection of protein tyrosine phosphatase interacting protein 51 (PTPIP51) to the nuclear factor κB (NFκB) signalling pathway. PTPIP51 mRNA and protein expression is regulated by RelA. If bound to the PTPIP51 promoter, RelA repress the mRNA and protein expression of PTPIP51. The parallel treatment with pyrrolidine dithiocarbamate (PDTC) reversed the suppression of PTPIP51 protein expression induced by TNFα. Using the intensity correlation analysis PTPIP51 verified a co-localization with RelA, which is also regulated by TNFα administration. Moreover, the direct interaction of PTPIP51 and RelA was established using the DuoLink proximity ligation assay. IκBα, the known inhibitor of RelA, also interacted with PTPIP51. This hints to the fact that in un-stimulated conditions PTPIP51 forms a complex with RelA and IκBα. The PTPIP51/RelA/IκBα complex is modulated by TNFα. Interestingly, the impact on the mitogen activated protein kinase pathway was negligible except in highest TNFα concentration. Here, PTPIP51 and Raf-1 interactions were slightly repressed. The newly established relationship of PTPIP51 and the NFκB signaling pathway provides the basis for a possible therapeutic impact.

The known interactome of PTPIP51 in HaCaT cells­inhibition of kinases and receptors.

Protein tyrosine phosphatse interacting protein (PTPIP51) is involved in the modulation of the mitogen activated protein kinase (MAPK) signaling pathway. Up to now, less is known about the regulation of this modulation. A recent study hinted to the phosphorylation status of PTPIP51 being essential for correct regulation of PTPIP51 function.In this study we investigate the phosphorylation status of PTPIP51 under the inhibition of the main interacting kinases and phosphatases of PTPIP51. c-Src was inhibited by Dasatinib, EGF receptor by Gefitinib, protein kinase C by staurosporine, protein kinase A by RpcAMPs and PTP1B by its specific inhibitor. Furthermore, a combination of PP2 with Gefitinib and RpcAMPs was used, respectively. The data were acquired for non-EGF and EGF treated HaCaT cells.All cells were analyzed relative to the subcellular distribution and change in the amount of tyrosine 176 phosphorylated PTPIP51. Furthermore, the protein interactions were assayed by duolink proximity ligation assay.HaCaT cells submitted to the respective inhibitor displayed a subcellular redistribution of tyrosine 176 phosphorylated PTPIP51 depending on the applied inhibitor. Yet, the amount of tyrosine 176 phosphorylated PTPIP51 remained unchanged by inhibitor treatment except for Gefitinib and simultaneous PP2 and Gefitnib treatment in non EGF-stimulated cells, but was elevated if cells were also EGF stimulated, in control and inhibitor treated cells. Interestingly, the interaction with EGFR, 14-3-3, Raf-1, c-Src, PTP1B, PKA and PKC was influenced by the application of inhibitors. Also EGF application resulted in a sharp drop of the PTPIP51 interaction with the MAPK pathway (e.g. Raf-1) in the control group.Summarizing these new findings, we postulate that PTPIP51 is regulated by its phosphorylation status combined with a thereby induced subcellular redistribution. In addition, the EGF receptor regulates PTPIP51 interaction with Raf-1 by its phosphorylation, thus preventing an overshooting activation of the MAPK pathway.

PTPIP51 in protein interactions: regulation and in situ interacting partners.

This study investigated the regulation of 14-3-3ß binding to PTPIP51 by the tyrosine phosphorylation status of PTPIP51. The tyrosine 176 residue is phosphorylated by c-Src. Up to now, nothing is known about the impact of such well-established phosphorylation events on the interaction profile of PTPIP51 with its partners of the mitogen-activated protein kinase (MAPK) pathway. In human keratinocytes the PTPIP51 phosphorylation was varied by inhibiting the phosphatase activity, thus enhancing the phosphorylation of PTPIP51. Differential blocking of Src kinase family members (despite c-Src) by PP2 increased the activity of c-Src and the tyrosine phosphorylation of PTPIP51 at position 176, which is the substrate of c-Src kinase. The amount of PTPIP51 interactions with 14-3-3ß, Raf-1, PTP1B and c-Src was evaluated and the resulting data were compared to an untreated control group. The increased phosphorylation level resulted in a sharp drop of the 14-3-3ß/PTPIP51 and 14-3-3ß/Raf-1 interaction. Besides the 14-3-3 interaction of PTPIP51, the interaction with the two MAPK modulators, protein kinase A (PKA) and diacylglycerol kinase alpha (DAGKα), are also regulated by the tyrosine phosphorylation status of PTPIP51. Additional immunostaining experiments were done investigating the functional implication on these interactions of the phosphorylation in apoptotic processes. In the pervanadate- and PP2-treated HaCaT cells, higher amounts of apoptotic cells were not detected as compared to the control group. The presented data confirms a tyrosine phosphorylation-dependent interaction of PTPIP51 with 14-3-3ß and Raf-1 in vivo and a tyrosine-dependent interaction profile with DAGKα and PKA. The non-interaction of PTPIP51 with 14-3-3 is not sufficient for triggering apoptosis.

Expression of PTPIP51 during mouse eye development.

The expression pattern of the novel tyrosine phosphatase interacting protein 51 (PTPIP51) was studied during the organogenesis of mouse eye on a transcriptional (RT-PCR and in situ hybridization) and translational level (immunohistochemistry and immunoblotting). Timed developmental stages from day E12 to day E18 were analyzed regarding the distribution of PTPIP51 and compared to the expression patterns observed during postnatal developmental stages and adult eye. PTPIP51 was found to be expressed in all investigated developmental stages in derivatives of mesoderm and ectoderm, such as developing cornea, lens, neuroretina and extraocular muscles. Conjuctiva and corneal epithelia were PTPIP51 reactive during all investigated developmental stages including the mature eye. Embryonic differentiation led to reactive keratocytes of the corneal stroma and remained so in post partal stages, as well as in the adult eye. On day E12, all cells comprising the developing lens body showed PTPIP51 expression. Further development unto the adult eye resulted in a restriction of PTPIP51 expression to the anterior lens epithelium and finally to the equatorial region of the lens epithelium. The developing neuroretina showed a strong PTPIP51 expression in the inner neuroblastic layer and the future receptor cell layer. In the adult eye, the retinal ganglion cells and the inner nuclear layer remained PTPIP51 reactive. The data presented here suggests PTPIP51 to be integrated in signaling cascades regulating differentiation and apoptosis during eye development.

Expression of the novel protein PTPIP51 in rat liver: an immunohistochemical study.

Expression of the novel protein tyrosine phosphatase interacting protein 51 (PTPIP51) was investigated on mRNA and protein level in the liver of adult Wistar rats. The presence of PTPIP51 mRNA was detected by Northern blotting. Immunostaining showed expression of PTPIP51 protein in distinct non-parenchymal cells. These cells were identified as Kupffer cells, stellate cells and natural killer cells by detection of cell-specific antigens. Whereas most endothelial cells lining large vessels reacted positive to the PTPIP51 antibody, sinusoidal endothelium showed no detectable amount of PTPIP51. Furthermore, PTPIP51 was also found to be expressed in cells forming the biliary tree. An additional subcellular analysis of the non-parenchymal cells by means of electron microscopy showed the presence of PTPIP51 protein in the cytoplasm and in the nuclei of non-parenchymal cells. Most of the hepatocytes did not show any immuno-detectable amount of PTPIP51, yet, some revealed PTPIP51 protein either in the cytoplasm or in the nucleus.

FRET-CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections.

It is pivotal to identify protein-protein interaction in situ to understand protein function. Conventional methods to determine the interaction of proteins destruct tissue or are applicable to cell culture only. To identify association of proteins in cells in tissue, we adapted indirect double-labeling immunofluorescence and combined it with conventional confocal laser scanning microscopy (CLSM) to measure fluorescence resonance energy transfer (FRET). As a model system, we chose caveolin-1alpha and caveolin-2, two major components of endothelial caveolae, and examined their interaction in the endothelium of vessels in fixed tissues of laboratory animals and human glomus tumors. Several methodological aspects were examined. Measuring the absolute increase in fluorescence (DeltaIF) was superior compared to determining the relative FRET efficiency, because it is more robust against small increases of fluorescence during measurements that results from unavoidable minimal crossreactivity of the secondary antibodies. Both, sequential and simultaneous incubation of secondary antibodies result in robust and reliable increases in DeltaIF. If incubated sequentially, however, the acceptor-labeled secondary antibody should be applied first. The size of the secondary reagent (F(ab')2 vs whole antibody) has no major influence. In conclusion, CLSM-FRET can measure close spatial association of proteins in situ and can be applied to human surgical material.

Epidermal growth factor-, transforming growth factor-beta-, retinoic acid- and 1,25-dihydroxyvitamin D3-regulated expression of the novel protein PTPIP51 in keratinocytes.

The novel protein PTPIP51 (protein tyrosine phosphatase-interacting protein 51), which has been found to interact with protein tyrosine phosphatases of the PTP1B/TcPTP subfamily, is expressed in all suprabasal layers of human epidermis. Hence, a human keratinocyte cell line (HaCaT) grown on culture slides was used as a simplified model system to study the influence of hormonal agents on the regulation of PTPIP51 expression. Results were obtained by immunocytochemistry and subsequent statistical analysis. Additionally, immunoblotting was performed to detect the possible occurrence of distinct molecular weight forms as described previously. Subcellular localization of PTPIP51 protein was analyzed by specific staining of cellular organelles. HaCaT cells were subjected to treatment with factors that are crucial for the regulation of proliferation and differentiation of keratinocytes in human epidermis: epidermal growth factor (EGF), transforming growth factor-beta(TGF-beta), retinoic acid (RA) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Epidermal growth factor receptor (EGFR) expressed in HaCaT cells was inhibited by PD153035. Only about 35% of untreated HaCaT cells were immunoreactive for the PTPIP51 protein. Whereas cells treated with increasing concentrations of 1,25 (OH)(2)D(3) showed a stepwise numerical increase of PTPIP51-positive cells, treatment with RA did not influence the number of PTPIP51-positive cells except when supraphysiological concentrations were applied. Concentration-dependent increase of cells stained positive for PTPIP51 was also observed when HaCaT cells were subjected to EGF treatment. Additional treatment of these cells with PD153035 led to a slight decrease in the fraction of PTPIP51-positive cells, which was not statistically significant. Immunoblotting results suggest a specific pattern of different molecular weight forms of PTPIP51 being expressed in HaCaT cells. Subcellular analysis revealed an association of the protein with mitochondria in nonconfluent cells, whereas confluent cells lack such correlation. The intracellular distribution of PTPIP51 resembled the localization of its interacting partner TcPTP. Furthermore, PTPIP51 was found to be present in both the nucleus and cytoplasm of HaCaT cells. In summary, the results indicate a possible association of PTPIP51 expression with differentiation as well as with apoptosis of keratinocytes.

The novel protein PTPIP51 exhibits tissue- and cell-specific expression.

The expression patterns of both mRNA and protein of the novel protein tyrosine phosphatase interacting protein 51 (PTPIP51) were studied in various organs by in situ hybridization, immunoblotting, and immunocytochemistry. The protein was found in all mammalian species investigated: guinea pig, rat, mouse, pig, and human. The presence of the protein was, however, restricted to specific organs. High levels of PTPIP51 were found in epidermis and seminiferous epithelium. The expression appears to be associated with distinct stages of differentiation. While basal cells in the epidermis and spermatogonia showed no perceptible amount of PTPIP51, keratinocytes of suprabasal layers and differentiating first-order spermatocytes up to spermatids exhibited high expression. In skeletal muscle, the presence of PTPIP51 was restricted to fibers of the fast twitch type. In surface epithelia containing ciliated cells, the protein was associated with the microtubular structures responsible for ciliary movement. Furthermore, specific structures of the central nervous system, for example, neurons of the hippocampal region, ganglion cells of the autonomic nervous system, and axons of the peripheral nervous system showed a distinct staining pattern with the antibody to PTPIP51. Our data suggest that PTPIP51 might be involved in the regulation of cellular processes associated with differentiation, movement, or cytoskeletal organization.