Extracellular calcium regulates HeLa cell morphology during adhesion to gelatin: Role of translocation and phosphorylation of cytosolic phospholipaseA(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 p resence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Add ition 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 different ial release of AA and subsequent formation of lamellipodia and filopodia du ring HeLa cell adhesion, cPLA(2) phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and sp reading in the presence of Ca2+, all cPLA(2) became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA(2) phosphorylatio n was lengthened to <4 min. Maximal translocation of cPLA(2) from cytosol t o membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughou t the duration of cell spreading. The amount of total cellular cPLA(2) tran slocated to the membrane in the presence of extracellular Ca2+ went from <2 0% for unspread cells to >95% for spread cells. Ln the absence of Ca2+ only 55-65% of the total cPLA(2) was translocated to the membrane during cell s preading. The decrease in the amount translocated could account for the com parable decrease in the amount of AA released by cells during spreading wit hout extracellular Ca2+. Although translocation of cPLA(2) from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA(2) was attachment depe ndent 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 underwen t a rapid phosphorylation upon early attachment followed by a dephosphoryla tion. Both rates were enhanced during cell spreading in the presence of ext racellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 c ompletely inhibited the attachment-dependent ERK2 phosphorylation but did n ot inhibit cell spreading, cPLA(2) phosphorylation, translocation, or AA re lease. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-ace tate) 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. Ln conclusi on, 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) phosphorylati on and the release of AA.