Vol. 9, Issue 12, 3429-3443, December 1998

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A₂

John R. Crawford,^* and Bruce S. Jacobson

Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003

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 Ca²⁺ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca²⁺. Addition of exogenous AA to cells spreading in the absence of extracellular Ca²⁺ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A₂ (cPLA₂) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA₂ phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca²⁺, all cPLA₂ 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 cPLA₂ phosphorylation was lengthened to <4 min. Maximal translocation of cPLA₂ from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca²⁺ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA₂ translocated to the membrane in the presence of extracellular Ca²⁺ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca²⁺ only 55-65% of the total cPLA₂ 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 Ca²⁺. Although translocation of cPLA₂ from cytosol to membrane was Ca²⁺ dependent, phosphorylation of cPLA₂ was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA₂, 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 Ca²⁺. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA₂ phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA₂ and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA₂ translocation and phosphorylation remained unaffected. In conclusion, although cPLA₂-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 cPLA₂ phosphorylation and the release of AA.