CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca2+-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

Enhanced expression of transferrin receptor 1 contributes to oncogenic signalling by sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell-surface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.

Purification and characterization of recombinant murine sulfamidase.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in the lysosomal enzyme sulfamidase, which is required for the degradation of heparan sulfate. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. A naturally occurring mouse model to MPS IIIA exhibits a similar disease progression to that observed in patients. Disease in the mice results from a base substitution at codon 31 in the sulfamidase gene, altering an aspartic acid to an asparagine (D31N). This aspartic 31 is involved in binding of the divalent metal ion needed for catalytic function, and as such reduces the specific activity of the enzyme to about 3% of that of wild-type. The mutant protein has decreased stability and shows increased degradation over a 24 h chase period when compared to wild-type mouse sulfamidase. Mouse sulfamidase that was purified using a two-step ion exchange procedure was shown to have similar kinetic properties to that of purified human sulfamidase. Recombinant murine sulfamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.