~p l f 5~"~, a protein tyrosine kinase (PTK) co-localized with integrtns in focal adhesion plaques, is known to transduce signals invoked in the regulation of cell adhesion and motility as well as the anchorage-independent growth of transformed cells. We investigated whether pp I 25F*K could be part of a signalling pathway that contributes to the progression of human prostate carcinoma (PCa). Up-regulation of pp I 25F*K expression, its activation by phosphoryiation on tyrosine and its association with paxillin and p 5 W were preferentially observed in PCa tissues from patients with metastases, whereas normal and hyperplastic prostates and localized PCa tissues showed undetectable or low levels of both FAK mRNA and protein and an absence of pp12SFAK signalling complexes. The increase in expression and activation of pp I 25FAK in metastatic PCa tissues was also corroborated by our findings in human PCa cell lines. Indeed, higher levels of ~p l 2 5~~and FAK mRNA were observed in highly tumorigenic PC-3 cells as was the presence of activated p~1 2 5 "~, as opposed to an inactive form in LNCaP cells, which have a lower tumorigenic ability. In addition, ~~1 2 5~~ formed signalling complexes with both paxillin and p5Wk in PC-3 cells as in metastatic PCa tissues. Together, our results show that an increase in FAK mRNA and protein, as well as pp 125FM activation and association with signalling proteins, correlates with progression and invasion in human PCa tissues and cells.8 1996 Wiley-Liss, Inc.The processes of tumour formation, invasion and metastasis represent complex genetic and epigenetic events that lead to cell transformation, with subsequent changes in adhesive properties that allow the transformed cells to migrate, gain access to the circulation and form metastatic colonies (Liottaet al., 1991). It is likely that some changes in adhesion occur at focal adhesion plaques which are cell/extracellular matrix (ECM) contact points containing integrin receptors, cytoskeletal components and intracellular signalling proteins such as focal adhesion kinase ( p~1 2 5~"~) , a non-receptor protein tyrosine kinase (PTK) (Clark and Brugge, 1995; Schaller et al., 1992). Evidence from several laboratories has shown that cell adhesion to the ECM through the integrin receptors leads to an increase in the tyrosine phosphorylation of pp12FAK and a subsequent stimulation of its activity (Juliano and Haskill, 1993). Phosphorylation of ~~1 2 . 5~~ is also increased in Rous sarcoma virus-transformed cells, as a result of the expression of ~p 6 0 ' ' -~~~ oncogene product, which is also a PTK (Guan and Shalloway, 1992). These changes lead to the disassembly of actin filaments and focal adhesions and, consequently, to a decrease in cell adhesion and an increase in anchorageindependent growth.As part of the integrin signalling pathway, ~~1 2 5~"~ has also been shown to be associated with various intracellular proteins in fibroblasts and transformed cells . Among them, paxillin, a cytoskeletal phosphotyrosine (pY)-containing protein invol...

A group of four similar proteins, BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, represent the major acidic proteins found in bovine seminal plasma (BSP). These proteins are secretory products of the seminal vesicles; they bind to spermatozoa upon ejaculation and could represent decapacitation factors. It has been shown that the glycosaminoglycans present in the female reproductive tract are involved in the capacitation of spermatozoa. Therefore, it was of interest to investigate whether BSP-A1, -A2, -A3, and -30-kDa proteins of bovine seminal fluid interact with heparin. Chromatography of alcohol precipitates of bovine seminal fluid on a heparin-Sepharose column resolved these proteins into three peaks. Peaks 1 and 2 (retarded proteins) were eluted upon extensive washing of the column with 0.05 M phosphate buffer, pH 7.4 (equilibrating buffer), and accounted for approximately 25% of the applied proteins. Proteins in peak 3 represented adsorbed proteins and were eluted with phosphate buffer containing 1 M NaCl. Proteins in each peak were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Peak 1 contained proteins with molecular weights ranging from 8 to 350 kDa, peak 2 contained a single protein with a molecular weight of 14 kDa, and peak 3 contained proteins with molecular weights of 15.5, 16, 25, and 30 kDa. The proteins in peak 3 were further resolved into unadsorbed (peak 4) and adsorbed (peak 5) proteins on a gelatin-Agarose column. Separation of the proteins of peak 3 and peak 5 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents followed by transfer to nitrocellulose and probing with antibodies against the previously well-characterized BSP proteins indicated the presence of BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesteryl sulfate is a normal constituent of human spermatozoa. The in vitro uptake of tritiated cholesteryl sulfate resulted in the labeling of all spermatozoa as demonstrated by light-microscope radioautography. The binding of the sterol sulfate was localized mainly in the head and midpiece. Radioautography at the level of the electron microscope revealed that the sterol sulfate is localized on the plasma membrane, mostly in the region of the acrosome. Further proof for this localization was obtained by selective dissolution of the plasma membrane and acrosome of the spermatozoa with low concentrations of Triton X-100. This treatment resulted in the simultaneous removal of tritiated cholesteryl sulfate bound to the spermatozoa. A hypothesis is presented concerning the role of cholesteryl sulfate as a membrane stabilizer and enzyme inhibitor during the maturation of spermatozoa in the epididymis. According to this hypothesis, the cleavage ofthe sulfate moiety within the female reproductive tract triggers a cascade of events leading to sperm capacitation and fertilization. One of the challenges remaining in reproductive biology is the elucidation ofthe biochemical events involved in the maturation and capacitation of mammalian spermatozoa. A review of the literature (1)(2)(3)(4)(5) indicates that the following biochemical events occur during capacitation. Stabilizing factor(s), associated with the spermatozoal membrane during transit or storage within the epididymis inhibit the release ofacrosomal enzymes in the male tract, but the factor(s) must be removed during migration in the female tract to allow the contact of the acrosomal enzymes with the investments of the ovum in order to facilitate penetration. Removal ofthe stabilizing substance(s) is thought to be enzymecatalyzed and involves changes in membrane conformation and permeability, ultimately leading to the acrosome reaction.Evidence is accumulating in support of our contention that sterol sulfates play an important role in the biochemistry of sperm maturation and capacitation. Thus, we have reported that cholesteryl sulfate (CholSO4) is an important component of human spermatozoa and is avidly taken up by these cells during in vitro incubation (6, 7). In addition, during transit through the epididymis, hamster spermatozoa exhibit a severalfold increase in desmosteryl sulfate concentration from the caput to the cauda regions (8). The finding of sterol sulfotransferase activity in the hamster epididymis (9) demonstrates that the biosynthesis of sterol sulfates can occur in this tissue.That low concentrations of sterol sulfates can block in vitro capacitation by hamster cumulus cells (10) and also inhibit acrosin (11), the sperm acrosomal proteinase involved in the penetration of the zona pellucida of the ovum (12), provides evidence that sterol-sulfatase (sterol-sulfate sulfohydrolase, EC 3.1.6.2) could be involved in the mechanism of sperm capacitation and ovum penetration. Furthermore, sterol-sulfatase is present in the human female reproduct...