Identification of a 68 kDa protein which copurifies with type-1 protein phosphatase as albumin

AbstractProteins of 60–70 kDa copurify with some preparations of type-1 or type-2 phosphatases. In our system chromatography on polylysine-Affi-Gel 10 separates a 68 kDa protein from rabbit muscle glycogen particle phosphorylase phosphatase. The separation affects neither the activity nor the size of the phosphatase. The 68 kDa protein, although pure by SDS gel electrophoresis criteria, still displays phosphatase activity of approx. 6–8 Umg. However, rechromatography either on Bio-Gel A-0.5 m or on Blue Sepharose CL-6B followed by gel filtration shows that the activity is due to a contamination with phosphatases of type 1 and type 2, displaying a molecular mass of 35 kDa, which can be totally removed from the 68 kDa protein. The amino acid composition of the 68 kDa protein is identical to that of rabbit serum albumin, within the limits of variation of the method. Furthermore, the sequence of the 38 N-terminal amino acids is the same in the isolated 68 kDa protein and in rabbit serum albumin

Non-hormonal treatment of vulvo-vaginal atrophy-related symptoms in post-menopausal women

In post-menopausal period vulvo-vaginal atrophy (VVA)-related symptoms may seriously affect women's quality of life. Hormonal replacement therapy effectively relieves these symptoms but it is not always safe or accepted, and a non-hormonal treatment is often needed instead. Over a period of 12 weeks, we tested the effect of a twice-a-week vulvo-vaginal application of a hyaluronic acid, AC collagen, isoflavones and vitamins-based cream (Perilei Pausa(®)) on 35 women in post-menopausal period, reporting VVA-related symptoms. After 12 weeks of treatment with Perilei Pausa(®) a significant improvement in vaginal dryness, vulvo-vaginal itching, dyspareunia (P < 0.001), dysuria (P = 0.02), nocturia (P = 0.009) and pollakiuria (P = 0.005) was reported by the women. Colposcopical score assessing the intensity of atrophic colpitis, cervico-vaginal paleness and petechiae was also reduced (P = 0.037, P = 0.016 and P = 0.032, respectively). No significant difference in terms of maturation value of cervico-vaginal epithelium was observed. In conclusion, Perilei Pausa(®) may represent an effective and safe alternative treatment of symptomatic VVA in post-menopausal women

Overexpression of leucocyte common antigen (LAR) P-subunit in thyroid carcinomas

Protein tyrosine phosphatase (PTPase) dephosphorylation and protein tyrosine kinase (PTKs) phosphorylation of key signal transduction proteins may be regulated by extracellular signals, making PTPases important in the regulation of cell proliferation. Leucocyte common antigen (LAR), a receptor-like PTPase, consists of E-subunit, containing the cell adhesion molecule-like receptor region, and P-subunit specific for a short segment of the extracellular region, the transmembrane peptide, and two cytoplasmic PTPase domains. We produced a monoclonal antibody against the LAR P-subunit for immunohistochemical screening of LAR expression in normal and tumourous tissues. Gliomas and gastric, colorectal, lung, breast and prostate cancers showed weak and relatively infrequent expression. Intense and diffuse expression, however, was detected in 95% (227 out of 239) of thyroid carcinomas, but only 12% (22 out of 128) of adenomas and no cases of benign thyroid disease were immunopositive. In contrast to broad staining in carcinomas, LAR expression in thyroid adenomas was often found in small focal or locally invasive areas. Western blot analysis similarly detected LAR P-subunit protein in thyroid carcinomas, but not in normal tissues. We believe this to be the first demonstration of LAR overexpression in thyroid carcinoma and may help to elucidate the role of PTPases in the development of malignancy

The extracellular-regulated protein kinase 5 (ERK5) enhances metastatic burden in triple-negative breast cancer through focal adhesion protein kinase (FAK)-mediated regulation of cell adhesion

From Springer Nature via Jisc Publications RouterHistory: received 2020-04-21, rev-recd 2021-03-23, accepted 2021-04-14, registration 2021-04-15, pub-electronic 2021-05-12, online 2021-05-12, pub-print 2021-06-10Publication status: PublishedFunder: Worldwide Cancer Research; doi: https://doi.org/10.13039/100011713; Grant(s): 15-1283Funder: RCUK | MRC | Medical Research Foundation; doi: https://doi.org/10.13039/501100009187; Grant(s): MC_PC_18056Abstract: There is overwhelming clinical evidence that the extracellular-regulated protein kinase 5 (ERK5) is significantly dysregulated in human breast cancer. However, there is no definite understanding of the requirement of ERK5 in tumor growth and metastasis due to very limited characterization of the pathway in disease models. In this study, we report that a high level of ERK5 is a predictive marker of metastatic breast cancer. Mechanistically, our in vitro data revealed that ERK5 was critical for maintaining the invasive capability of triple-negative breast cancer (TNBC) cells through focal adhesion protein kinase (FAK) activation. Specifically, we found that phosphorylation of FAK at Tyr397 was controlled by a kinase-independent function of ERK5. Accordingly, silencing ERK5 in mammary tumor grafts impaired FAK phosphorylation at Tyr397 and suppressed TNBC cell metastasis to the lung without preventing tumor growth. Collectively, these results establish a functional relationship between ERK5 and FAK signaling in promoting malignancy. Thus, targeting the oncogenic ERK5-FAK axis represents a promising therapeutic strategy for breast cancer exhibiting aggressive clinical behavior

Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice.

Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1