Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts.

Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co-cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild-type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild-type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.

Ppp2r2a Knockout Mice Reveal That Protein Phosphatase 2A Regulatory Subunit, PP2A-B55α, Is an Essential Regulator of Neuronal and Epidermal Embryonic Development.

The serine/threonine protein phosphatase 2A (PP2A) is a master regulator of the complex cellular signaling that occurs during all stages of mammalian development. PP2A is composed of a catalytic, a structural, and regulatory subunit, for which there are multiple isoforms. The association of specific regulatory subunits determines substrate specificity and localization of phosphatase activity, however, the precise role of each regulatory subunit in development is not known. Here we report the generation of the first knockout mouse for the Ppp2r2a gene, encoding the PP2A-B55α regulatory subunit, using CRISPR/Cas9. Heterozygous animals developed and grew as normal, however, homozygous knockout mice were not viable. Analysis of embryos at different developmental stages found a normal Mendelian ratio of Ppp2r2a-/- embryos at embryonic day (E) 10.5 (25%), but reduced Ppp2r2a-/- embryos at E14.5 (18%), and further reduced at E18.5 (10%). No live Ppp2r2a-/- pups were observed at birth. Ppp2r2a-/- embryos were significantly smaller than wild-type or heterozygous littermates and displayed a variety of neural defects such as exencephaly, spina bifida, and cranial vault collapse, as well as syndactyly and severe epidermal defects; all processes driven by growth and differentiation of the ectoderm. Ppp2r2a-/- embryos had incomplete epidermal barrier acquisition, associated with thin, poorly differentiated stratified epithelium with weak attachment to the underlying dermis. The basal keratinocytes in Ppp2r2a-/- embryos were highly disorganized, with reduced immunolabeling of integrins and basement membrane proteins, suggesting impaired focal adhesion and hemidesmosome assembly. The spinous and granular layers were thinner in the Ppp2r2a-/- embryos, with aberrant expression of adherens and tight junction associated proteins. The overlying stratum corneum was either absent or incomplete. Thus PP2A-B55α is an essential regulator of epidermal stratification, and is essential for ectodermal development during embryogenesis.

Functional importance of PP2A regulatory subunit loss in breast cancer.

PURPOSE: Protein phosphatase 2A (PP2A) is a family of serine/threonine phosphatases that regulate multiple cellular signalling pathways involved in proliferation, survival and apoptosis. PP2A inhibition occurs in many cancers and is considered a tumour suppressor. Deletion/downregulation of PP2A genes has been observed in breast tumours, but the functional role of PP2A subunit loss in breast cancer has not been investigated. METHODS: PP2A subunit expression was examined by immunohistochemistry in human breast tumours, and by qPCR and immunoblotting in breast cancer cell lines. PP2A subunits were inhibited by shRNA, and mutant PP2A genes overexpressed, in MCF10A and MCF7 cells, and growth and signalling in standard and three-dimensional cultures were assessed. RESULTS: Expression of PP2A-Aα, PP2A-Bα and PP2A-B'α subunits was significantly lower in primary human breast tumours and lymph node metastases, compared to normal mammary tissue. PP2A-Aα and the regulatory subunits PP2A-Bα, -Bδ and -B'γ were also reduced in breast cancer cell lines compared to normal mammary epithelial cells. Functionally, shRNA-mediated knockdown of PP2A-Bα, -B'α and -B'γ, but not PP2A-Aα, induced hyper-proliferation and large multilobular acini in MCF10A 3D cultures, characterised by activation of ERK. Expression of a breast cancer-associated PP2A-A mutant, PP2A-Aα-E64G, which inhibits binding of regulatory subunits to the PP2A core, induced a similar hyper-proliferative phenotype. Knockdown of PP2A-Bα also induced hyper-proliferation in MCF7 breast cancer cells. CONCLUSION: These results suggest that loss of specific PP2A regulatory subunits is functionally important in breast tumourigenesis, and support strategies to enhance PP2A activity as a therapeutic approach in breast cancer.

Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors.

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.