BRK phosphorylates SMAD4 for proteasomal degradation and inhibits tumor suppressor FRK to control SNAIL, SLUG, and metastatic potential.

The tumor-suppressing function of SMAD4 is frequently subverted during mammary tumorigenesis, leading to cancer growth, invasion, and metastasis. A long-standing concept is that SMAD4 is not regulated by phosphorylation but ubiquitination. Our search for signaling pathways regulated by breast tumor kinase (BRK), a nonreceptor protein tyrosine kinase that is up-regulated in ~80% of invasive ductal breast tumors, led us to find that BRK competitively binds and phosphorylates SMAD4 and regulates transforming growth factor-ß/SMAD4 signaling pathway. A constitutively active BRK (BRK-Y447F) phosphorylates SMAD4, resulting in its recognition by the ubiquitin-proteasome system, which accelerates SMAD4 degradation. Activated BRK-mediated degradation of SMAD4 is associated with the repression of tumor suppressor gene FRK and increased expression of mesenchymal markers, SNAIL, and SLUG. Thus, our data suggest that combination therapies targeting activated BRK signaling may have synergized the benefits in the treatment of SMAD4 repressed cancers.

Constitutive activation of breast tumor kinase accelerates cell migration and tumor growth in vivo.

Breast tumor kinase (BRK) is a non-receptor tyrosine kinase overexpressed in most human breast tumors, including lymph node metastases, but undetected in normal mammary tissue or in fibroadenomas. The activity of BRK-like Src family tyrosine kinase, is regulated negatively by phosphorylation of C-terminal tyrosine 447. Although the kinase that regulates BRK activation has not been identified, we and others have previously shown that BRK-Y447F is a constitutively active variant. Because BRK-Y447F significantly enhances the catalytic activity of the enzyme, we investigated the role of the constitutively active BRK variant in tumor formation and metastasis. Using stable breast cancer cell MDA-MB-231 we observed significantly enhanced rates of cell proliferation, migration and tumor formation in BRK-Y447F stable cells compared with wild-type stable cell lines. Our results indicate full activation of BRK is an essential component in the tumorigenic role of BRK.

Sam68 haploinsufficiency delays onset of mammary tumorigenesis and metastasis.

The Src-associated substrate in mitosis Sam68 is a KH type RNA-binding protein known to be a substrate of numerous tyrosine kinases, and often referred to as a STAR (signal transduction activator of RNA) protein. Herein, we observed that Sam68-null mice display mammary gland and the uterine development defects. Moreover, we report that Sam68 haploinsufficiency impedes mammary tumor onset in vivo driven by the potent mammary-targeted polyoma middle T-antigen (MMTV-PyMT) oncogene. The effect was cell autonomous as the Sam68 knockdown in PyMT-transformed cell lines also delayed tumorigenesis and metastasis formation in nude mice. Interestingly, tumor extracts isolated from PyMT/Sam68(+/-) mice compared with PyMT/Sam68(+/+) mice contained activated Src and FAK kinases. These findings suggest that Sam68 may be a modulator of tyrosine kinase activity in vivo and a signaling requirement for mammary tumorigenesis and metastasis.

Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail.

Sialidase (neuraminidase), encoded by the neu-1 gene in the major histocompatibility complex locus catalyzes the intralysosomal degradation of sialylated glycoconjugates. Inherited deficiency of sialidase results in sialidosis or galactosialidosis, both severe metabolic disorders associated with lysosomal storage of oligosaccharides and glycopeptides. Sialidase also plays an important role in cellular signaling and is specifically required for the production of cytokine interleukin-4 by activated T lymphocytes. In these cells, neu-1-encoded sialidase activity is increased on the cell surface, suggesting that a specific mechanism regulates sorting of this enzyme to the plasma membrane. We investigated that mechanism by first showing that sialidase contains the internalization signal found in lysosomal membrane proteins targeted to endosomes via clathrin-coated pits. The signal consists of a C-terminal tetrapeptide (412)YGTL(415), with Tyr(412) and Leu(415) essential for endocytosis of the enzyme. We further demonstrated that redistribution of sialidase from lysosomes to the cell surface of activated lymphocytes is accompanied by increased reactivity of the enzyme with anti-phosphotyrosine antibodies. We speculate that phosphorylation of Tyr(412) results in inhibition of sialidase internalization in activated lymphocytes.

Mutations in sialidosis impair sialidase binding to the lysosomal multienzyme complex.

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the catabolism of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots, and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation, and hepatosplenomegaly (sialidosis type II). We analyzed the effect of the missense mutations G68V, S182G, G227R, F260Y, L270F, A298V, G328S, and L363P, which are identified in the sialidosis type I and sialidosis type II patients, on the activity, stability, and intracellular distribution of sialidase. We found that three mutations, F260Y, L270F, and A298V, which are clustered in the same region on the surface of the sialidase molecule, dramatically reduced the enzyme activity and caused a rapid intralysosomal degradation of the expressed protein. We suggested that this region might be involved in sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in the multienzyme lysosomal complex required for the expression of sialidase activity. Transgenic expression of mutants followed by density gradient centrifugation of cellular extracts confirmed this hypothesis and showed that sialidase deficiency in some sialidosis patients results from disruption of the lysosomal multienzyme complex.

Clinical presentation of congenital sialidosis in a patient with a neuraminidase gene frameshift mutation.

UNLABELLED: Congenital sialidosis is a rare lysosomal storage disease caused by a primary neuraminidase deficiency which results from defects in the neuraminidase gene on chromosome 6p. The inheritance is autosomal recessive. Patients exhibit excessive urinary excretion of bound sialic acid and decreased or undetectable amounts of neuraminidase activity in various tissues. The clinical expression is variable, but ascites and hepatosplenomegaly are hallmarks of the disease. Skeletal abnormalities, facial dysmorphism and inguinal herniae have been described in most of the few reported cases. We describe a baby girl with biochemically proven sialidosis, who in addition to the above clinical features, had severely dilated coronary arteries, excessive retinal vascular tortuosity and an erythematous, macular rash. Homozygosity for a frameshift mutation at residue 623 of the neuraminidase cDNA was found. We speculate that the additional features found in our patient might be associated with the here described genotype of congenital sialidosis. CONCLUSION: Severely dilated coronary arteries, excessive retinal vascular tortuosity and an erythematous macular rash might be associated features of congenital sialidosis.

Molecular and structural studies of Japanese patients with sialidosis type 1.

To gain insight into the pathogenesis of sialidosis type 1, we performed molecular investigations of two unrelated Japanese patients. Both of them are compound heterozygotes for base substitutions of 649G-to-A and 727G-to-A, which result in amino acid alterations V217M and G243R, respectively. Using homology modeling, the structure of human lysosomal neuraminidase was constructed and the structural changes caused by these missense mutations were deduced. The predicted change due to V217M was smaller than that caused by G243R, the latter resulting in a drastic, widespread alteration. The overexpressed gene products containing these mutations had the same molecular weight as that of the wild type, although the amounts of the products were moderately decreased. A biochemical study demonstrated that the expressed neuraminidase containing a V217M mutation was partly transported to lysosomes and showed residual enzyme activity, although a G243R mutant was retained in the endoplasmic reticulum/Golgi area and had completely lost the enzyme activity. Considering the data, we surmise that the V217M substitution may be closely associated with the phenotype of sialidosis type 1 with a late onset and moderate clinical course.

Characterization of the sialidase molecular defects in sialidosis patients suggests the structural organization of the lysosomal multienzyme complex.

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation and hepatosplenomegaly (sialidosis type II). We have analyzed the genomic DNA from nine sialidosis patients of multiple ethnic origin in order to find mutations responsible for the enzyme deficiency. The activity of the identified variants was studied by transgenic expression. One patient had a frameshift mutation (G623delG deletion), which introduced a stop codon, truncating 113 amino acids. All others had missense mutations: G679G-->A (Gly227Arg), C893C-->T (Ala298Val), G203G-->T (Gly68Val), A544A-->G (Ser182Gly) C808C-->T (Leu270Phe) and G982G-->A (Gly328Ser). We have modeled the three-dimensional structure of sialidase based on the atomic coordinates of the homologous bacterial sialidases, located the positions of mutations and estimated their potential effect. This analysis showed that five mutations are clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We hypothesize that this region may be involved in the interface of sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in their high-molecular-weight complex required for the expression of sialidase activity in the lysosome.

Identification of UDP-N-acetylglucosamine-phosphotransferase-binding sites on the lysosomal proteases, cathepsins A, B, and D.

A key step in the targeting of soluble lysosomal enzymes is their recognition and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucosamine-phosphotransferase (phosphotransferase). The molecular mechanism of recognition is still unknown, but previous experiments suggested that the phosphotransferase-binding sites on lysosomal proteins are represented by structurally conserved surface patches of amino acids. We identified four such regions on nonhomologous lysosomal enzymes, cathepsins A, B, and D, which were superimposed by rotating their structures around the Calpha atom of the glycosylated Asn residue. We proposed that these regions represent putative phosphotransferase-binding sites and tested synthetic peptides, derived from these regions on the basis of surface accessibility, for their ability to inhibit in vitro phosphorylation of purified cathepsins A, B, and D. Our results indicate that cathepsin A and cathepsin D have one closely related phosphotransferase recognition site represented by a structurally and topologically conserved beta-hairpin loop, similar to that previously identified in lysosomal beta-glucuronidase. The most potent inhibition of phosphorylation was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosphorylated by the phosphotransferase. We propose that recognition and catalytic sites of the phosphotransferase are located on different subunits, therefore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.

Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation.

Galactosialidosis is an inherited lysosomal storage disease caused by the combined deficiency of lysosomal sialidase and beta-galactosidase secondary to the deficiency of cathepsin A/protective protein, which is associated with sialidase and beta-galactosidase in a high-molecular weight (1.27MDa) complex. Clinical phenotypes of patients as well as the composition of compounds which are stored in patient's tissues implicate sialidase deficiency as the underlying pathogenic defect. The recent cloning and sequencing of lysosomal sialidase [Pshezhetsky, Richard, Michaud, Igdoura, Wang, Elsliger, Qu, Leclerc, Gravel, Dallaire and Potier (1997), Nature Genet. 15, 316-320] allowed us to study the molecular mechanism of sialidase deficiency in galactosialidosis. By Western blotting, using antibodies against the recombinant human enzyme, and by NH2-terminal sequencing, we showed that sialidase is synthesized as a 45.5 kDa precursor and after the cleavage of the 47-amino acid signal peptide and glycosylation becomes a 48.3 kDa mature active enzyme present in the 1.27 kDa complex. Transgenic expression of sialidase in cultured skin fibroblasts from normal controls and from galactosialidosis patients, followed by immunofluorescent and immunoelectron microscopy showed that in both normal and affected cells the expressed sialidase was localized on lysosomal and plasma membranes, but the amount of sialidase found in galactosialidosis cells was approximately 5-fold reduced. Metabolic labelling studies demonstrated that the 48.3 kDa mature active form of sialidase was stable in normal fibroblasts (half-life approximately 2.7 h), whereas in galactosialidosis fibroblasts the enzyme was rapidly converted (half-life approximately 30 min) into 38.7 and 24 kDa catalytically inactive forms. Altogether our data provide evidence that the molecular mechanism of sialidase deficiency in galactosialidosis is associated with abnormal proteolytic cleavage and fast degradation.