Regulation of sphingosine kinase 1 signalling by calcium- and integrin-binding proteins.

Abstract

Sphingosine kinase 1 (SK1) catalyses the conversion of sphingosine to sphingosine-1-phosphate (S1P). Since elevated levels of cellular S1P have a well characterised prosurvival, pro-proliferative and oncogenic effect in cells, the regulation of SK1 activity is the subject of much current focus. Cells typically have low basal levels of SK1 activity, which appears to have a 'housekeeping’ role in the sphingomyelin cycle. However, the catalytic activity of this enzyme can be increased by a number of growth factors, cytokines and other agonists, generating a greater pool of S1P, which then appears to be involved in its cellular signalling. The Pitson group have previously demonstrated that the agonist-induced activation of human SK1 is mediated through phosphorylation of this enzyme at Ser²²⁵. Following this phosphorylation, SK1 translocates from the cytosol to the plasma membrane, with both of these events critical for the pro-survival, anti-apoptotic and oncogenic effects of this enzyme. Prior to the current study, the mechanism for the rapid, agonist-induced translocation of SK1 to the plasma membrane remained undetermined. Previous studies have demonstrated the requirement of the calmodulin binding site in SK1 for this translocation event, however a direct requirement for calmodulin itself has yet to be shown. In this study, we show that calcium- and integrin-binding protein 1 (CIB1), a calmodulinlike molecule, mediates the agonist-induced translocation of SK1 to the plasma membrane. We also demonstrate the ability of CIB1 to act as calcium-myristoyl switch, providing a functional mechanism by which it can mediate SK1 localisation to the plasma membrane. In addition, CIB1 was shown to be critical for the agonist-induced production of S1P and also the anti-apoptotic signalling associated with SK1. Furthermore, CIB1 itself was shown to be potentially oncogenic, and a dominant-negative version of CIB1 was also able to inhibit H-Ras-induced oncogenesis. These studies have also investigated the three other members of the CIB family of proteins, CIB2, CIB3 and CIB4 and their roles in regulating SK1 localisation and subsequent signalling events. While CIB1 was critical for the agonist-induced translocation of SK1 to the plasma membrane, CIB2 acted in an opposite manner, blocking the translocation of this enzyme. Furthermore, expression of CIB2 enhanced cellular apoptosis, presumably through its inhibitory effects on the SK1 survival pathway. In addition, CIB2 blocked H-Ras mediated oncogenesis. Hence, CIB1 and CIB2 appear to have opposing roles in the cell in relation to the regulation of SK1 signalling. CIB3 and CIB4 also appeared to interact with SK1 in vitro, however the cellular function of these interactions was not elucidated in this study. Interestingly, despite having considerable sequence similarity to CIB1 and CIB2, neither protein acted in a similar manner to either CIB1 or CIB2 with respect to SK1 function. While CIB3 did not appear to have any role in the SK1 signalling pathway, studies suggested CIB4 likely to have additional binding partners in the cell masking the ability to determine the biological function of the CIB4-SK1 interaction. Overall, this study identified both the translocation mechanism of SK1, in addition to a natural suppressor of this event. As the localisation of SK1 at the plasma membrane is critical in the oncogenic signalling of this enzyme, these findings may represent potential new directions for anti-cancer therapeutics.