Role of Membrane Cholesterol Levels in Activation of Lyn upon Cell Detachment

Abstract

Cholesterol, a major component of the plasma membrane, determines the physicalproperties of biological membranes and plays a critical role in the assembly of membranemicrodomains. Enrichment or deprivation of membrane cholesterol affects the activities of manysignaling molecules at the plasma membrane. Cell detachment changes the structure of the plasmamembrane and influences the localizations of lipids, including cholesterol. Recent studies showedthat cell detachment changes the activities of a variety of signaling molecules. We previously reportedthat the localization and the function of the Src-family kinase Lyn are critically regulated by its membrane anchorage through lipid modifications. More recently, we found that the localization andthe activity of Lyn were changed upon cell detachment, although the manners of which vary betweencell types. In this review, we highlight the changes in the localization of Lyn and a role of cholesterolin the regulation of Lyn’s activation following cell detachment.

Keywords: Lyn activation; Src-family kinases; cell detachment; cell–scaffold interactions; cholesterol; membrane distribution; subcellular localization.

Figure 1

Trafficking of Src-family kinases. (a) The black arrow indicates biosynthetic trafficking of Lyn kinase. Distributions of c-Src and Fyn differ from that of Lyn. The dashed arrows indicate the translocation of c-Src between the plasma membrane and endosomes. The red and blue wavy lines represent myristic acids and palmitic acids, respectively; (b) Loss of cell–scaffold interactions did not internalize Lyn from the plasma membrane in HeLa S3 cells but internalizes c-Src and Fyn in NIH3T3 cells, whereas caveolin and cholesterol were internalized from the plasma membrane after cell detachment in both cell lines. However, loss of cell–cell interactions is capable of internalizing Lyn in MDCK cells.

Figure 2

Fractionations of cellular membranes. (a) Detergent-based fractionation. An ideal membrane fragment consists of detergent-insoluble portion (left half) and detergent-soluble portion (right half). The proteins in detergent-soluble membranes are solubilized with an appropriate concentration of detergent and separated by density gradient fractionation. (b) (i) Membrane fragments comprising differential ratios of proteins to lipids were separated by density gradient fractionation; (ii) the distribution pattern of a given protein of interest in density gradient fractionation (green area) reflects the density of the membrane segments harboring the protein of interest (green circle).

Figure 3

Cell detachment activates Lyn through cholesterol depletion. Cell detachment causes internalization of some lipid raft-related molecules, including caveolin and cholesterol, in the plasma membrane. Cholesterol depletion affects the characteristics of the membrane segments harboring Lyn, which is associated with activation of Lyn in suspended cells.