Sphingomyelin plays complex structural and signaling functions in the plasma membrane. Of special interest is that hydrolysis of sphingomyelin to ceramide can modulate dynamics of membrane rafts, which serve as signaling platforms for various receptors. This review is focused on a recently discovered sphingomyelin-binding protein, lysenin, which can be used as a unique probe to trace distribution and turnover of sphingomyelin in cellular membranes. We analyze the primary and secondary structures of lysenin with respect to its interaction with the plasma membrane. The speci¢city of lysenin binding to sphingomyelin, revealed by both biochemical and cytochemical approaches, is discussed.

We found that FccRII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P 2 and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase Ia (PIP5-kinase Ia) that colocalized with PI(4,5)P 2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C 374-440 fragment of PIP5-kinase Ia or the pleckstrin homology domain of phospholipase Cd 1 (PLCd 1 -PH), two probes binding PI(4,5)P 2 . These probes reduced the amount of PI(4,5)P 2 in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P 2 elevation during phagocytosis. Simultaneously, PLCd 1 -PH-GFP reduced the amount of PIP5-kinase Ia associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase Ia-GST bound PI(4,5)P 2 , phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLCd 1 -PH domain, and possibly also the C 374-440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase Ia for PI(4,5)P 2 binding in the plasma membrane leading eventually to PI(4,5)P 2 depletion.

Evaluation of the secondary structure of caldesmon from c.d. spectra revealed that it contains 51% helix, 9% beta-strand and 40% of remainder structures. These values agree well with the predicted ones from amino acid sequence, assuming an extended chain structure for caldesmon. The estimates of the secondary-structure elements in C-terminal 34 kDa and 19 kDa fragments are: 11 and 12% helix, 22 and 20% beta-strand, 13 and 17% beta-turns and loops, and 54 and 50% of remainder structure respectively. The best fit of experimental data was obtained assuming the globular state of the fragments. On the basis of structural analysis and fragmentation by proteolytic and chemical cleavages the six-domain model of caldesmon is proposed.

The interaction of caldesmon with liposomes composed of various phospholipids has been examined by tryptophan fluorescence spectroscopy. The results indicate that caldesmon makes its strongest complex with phosphatidylserine (PS) vesicles (Kass. = 1.45 x 10(5) M-1). Both electrostatic and hydrophobic interactions contribute to the stability of this complex. The site for strong binding of PS seems to be located in the N-terminal part of the 34 kDa C-terminal fragment of caldesmon. Binding of PS at this site results in displacement of calmodulin from its complex with caldesmon.

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