Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A(2)

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca(2+) released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca(2+). Addition of exogenous AA to cells spreading in the absence of extracellular Ca(2+) restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A(2) (cPLA(2)) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA(2) phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca(2+), all cPLA(2) became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca(2+), the time for complete cPLA(2) phosphorylation was lengthened to <4 min. Maximal translocation of cPLA(2) from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca(2+) and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA(2) translocated to the membrane in the presence of extracellular Ca(2+) went from <20% for unspread cells to >95% for spread cells. In the absence of Ca(2+) only 55–65% of the total cPLA(2) was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca(2+). Although translocation of cPLA(2) from cytosol to membrane was Ca(2+) dependent, phosphorylation of cPLA(2) was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA(2), the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca(2+). Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA(2) phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA(2) and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA(2) translocation and phosphorylation remained unaffected. In conclusion, although cPLA(2)-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA(2) phosphorylation and the release of AA.