Aberrant post-translational modifications in endosomal trafficking are potential therapeutic targets to avert therapy resistance in solid cancers: Dysregulation of PTM-regulated endosomal interactions presents an opportunity to block oncogenic signalling from multiple receptors by targeting common trafficking pathways: Dysregulation of PTM-regulated endosomal interactions presents an opportunity to block oncogenic signalling from multiple receptors by targeting common trafficking pathways.

Drugs targeting a single TK/RTK in the treatment of solid cancers has not had the same success seen in blood cancers. This is, in part, due to acquired resistance in solid cancers arising from a range of mechanisms including the upregulation of compensatory RTK signalling. Rather than attempting to inhibit individual compensatory RTK-requiring knowledge of which RTKs are upregulated in any given tumour-strategies to universally inhibit signalling from multiple RTKs may represent an effective alternative. Endosomal trafficking of RTKs is a common conduit that can regulate signalling from multiple RTKs simultaneously. As such, we posit that targeting endosomal trafficking-in particular, aberrant post-translational modifications in cancers that contribute to dysregulated endosomal trafficking-could inhibit oncogenic signalling driven by multiple RTKs and pave the way for the development of a novel class of inhibitors that shift the trafficking of RTKs to inhibit tumour growth.

PTPN14 phosphatase and YAP promote TGFß signalling in rheumatoid synoviocytes.

OBJECTIVE: We aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Gene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis. RESULTS: RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFß-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFß-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity. CONCLUSION: In RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.

Phosphorylation of serine 779 in fibroblast growth factor receptor 1 and 2 by protein kinase C(epsilon) regulates Ras/mitogen-activated protein kinase signaling and neuronal differentiation.

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser(779) in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser(779) in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser(779) was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCε can phosphorylate Ser(779) in vitro, whereas overexpression of PKCε results in constitutive Ser(779) phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCε reduces both growth factor-induced Ser(779) phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser(779), can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

Response to BMP4 signalling during ES cell differentiation defines intermediates of the ectoderm lineage.

The formation and differentiation of multipotent precursors underlies the generation of cell diversity during mammalian development. Recognition and analysis of these transient cell populations has been hampered by technical difficulties in accessing them in vivo. In vitro model systems, based on the differentiation of embryonic stem (ES) cells, provide an alternative means of identifying and characterizing these populations. Using a previously established mouse ES-cell-based system that recapitulates the development of the ectoderm lineage we have identified a transient population that is consistent with definitive ectoderm. This previously unidentified progenitor occurs as a temporally discrete population during ES cell differentiation, and differs from the preceding and succeeding populations in gene expression and differentiation potential, with the unique ability to form surface ectoderm in response to BMP4 signalling.

Quantifying EGFR endosomal recycling via immunofluorescence in breast cancer cells.

Previously published protocols for quantification of endosomal recycling are limited by the use of radioactive reagents, washing of cells in reducing buffers, or the requirement for large numbers of cells. Here, we describe a protocol for quantification of endosomal recycling using immunofluorescence that is optimized for EGFR in BT-549 breast cancer cells but could be applied to other RTKs and cell lines. Our protocol enables quick assessment of recycling and uses a relatively small number of cells. For complete details on the use and execution of this protocol, please refer to Lonic et al. (2021).

The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells.

There is an increasing appreciation that amino acids can act as signaling molecules in the regulation of cellular processes through modulation of intracellular cell signaling pathways. In culture, embryonic stem (ES) cells can be differentiated to a second, pluripotent cell population, early primitive ectoderm-like cells in response to biological activities within the conditioned medium MEDII. The amino acid l-proline has been identified as a component of MEDII required for ES cell differentiation. Here, we define the primary l-proline transporter on ES and early primitive ectoderm-like cells as sodium-coupled neutral amino acid transporter 2 (SNAT2). SNAT2 uptake of l-proline can be inhibited by the addition of millimolar concentrations of other substrates. The addition of excess amino acids was used to regulate the uptake of l-proline by ES cells, and the effect on differentiation was analyzed. The ability of SNAT2 substrates, but not other amino acids, to prevent changes in morphology, gene expression, and differentiation kinetics suggested that l-proline uptake through SNAT2 was required for ES cell differentiation. These data reveal an unexpected role for amino acid uptake and the amino acid transporter SNAT2 in regulation of pluripotent cells in culture and provides a number of specific, inexpensive, and nontoxic culture additives with the potential to improve the quality of ES cell culture.

Phosphorylation of PKCδ by FER tips the balance from EGFR degradation to recycling.

Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in ∼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.

L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture.

The development of cell therapeutics from embryonic stem (ES) cells will require technologies that direct cell differentiation to specific somatic cell lineages in response to defined factors. The initial step in formation of the somatic lineages from ES cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response to a biological activity contained within the conditioned medium MEDII. Fractionation of MEDII has identified two activities required for EPL cell formation, an activity with a molecular mass of <3 kDa and a second, much larger species. Here, we have identified the low-molecular-weight activity as l-proline. An inhibitor of l-proline uptake, glycine, prevented the differentiation of ES cells in response to MEDII. Supplementation of the culture medium of ES cells with >100 M l-proline and some l-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with l-proline since other amino acids and analogs of proline did not exhibit an equivalent activity. Activation of the mammalian target of rapamycin (mTOR) signaling pathway was found to be necessary but not sufficient for l-proline activity; addition of other activators of the mTOR signaling pathway failed to alter the ES cell phenotype. This is the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identifies a novel role for the imino acid l-proline.

The tyrosine phosphatase PTPN14 (Pez) inhibits metastasis by altering protein trafficking.

Factors secreted by tumor cells shape the local microenvironment to promote invasion and metastasis, as well as condition the premetastatic niche to enable secondary-site colonization and growth. In addition to this secretome, tumor cells have increased abundance of growth-promoting receptors at the cell surface. We found that the tyrosine phosphatase PTPN14 (also called Pez, which is mutated in various cancers) suppressed metastasis by reducing intracellular protein trafficking through the secretory pathway. Knocking down PTPN14 in tumor cells or injecting the peritoneum of mice with conditioned medium from PTPN14-deficient cell cultures promoted the growth and metastasis of breast cancer xenografts. Loss of catalytically functional PTPN14 increased the secretion of growth factors and cytokines, such as IL-8 (interleukin-8), and increased the abundance of EGFR (epidermal growth factor receptor) at the cell surface of breast cancer cells and of FLT4 (vascular endothelial growth factor receptor 3) at the cell surface of primary lymphatic endothelial cells. We identified RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-δ) as binding partners and substrates of PTPN14. Similar to cells overexpressing PTPN14, receptor trafficking to the cell surface was inhibited in cells that lacked PRKCD or RIN1 or expressed a nonphosphorylatable RIN1 mutant, and cytokine secretion was decreased in cells treated with PRKCD inhibitors. Invasive breast cancer tissue had decreased expression of PTPN14, and patient survival was worse when tumors had increased expression of the genes encoding RIN1 or PRKCD. Thus, PTPN14 prevents metastasis by restricting the trafficking of both soluble and membrane-bound proteins.

Fibroblast growth factor receptor 2 phosphorylation on serine 779 couples to 14-3-3 and regulates cell survival and proliferation.

The fibroblast growth factors (FGFs) exert their diverse (or pleiotropic) biological responses through the binding and activation of specific cell surface receptors (FGFRs). While FGFRs are known to initiate intracellular signaling through receptor tyrosine phosphorylation, the precise mechanisms by which the FGFRs regulate pleiotropic biological responses remain unclear. We now identify a new mechanism by which FGFR2 is able to regulate intracellular signaling and cellular responses. We show that FGFR2 is phosphorylated on serine 779 (S779) in response to FGF2. S779, which lies adjacent to the phospholipase Cgamma binding site at Y766, provides a docking site for the 14-3-3 phosphoserine-binding proteins and is essential for the full activation of the phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase pathways. Furthermore, S779 signaling is essential for promoting cell survival and proliferation in both Ba/F3 cells and BALB/c 3T3 fibroblasts. This new mode of FGFR2 phosphoserine signaling via the 14-3-3 proteins may provide an increased repertoire of signaling outputs to allow the regulation of pleiotropic biological responses. In this regard, we have identified conserved putative phosphotyrosine/phosphoserine motifs in the cytoplasmic domains of diverse cell surface receptors, suggesting that they may perform important functional roles beyond the FGFRs.