Regulation of actin cytoskeleton in lymphocytes: PKC-delta disrupts IL-3-induced membrane ruffles downstream of Rac1

In the murine pre-B lymphoid cell line Baf3, the presence of IL-3 is requir ed for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL-3 disso lves ruffled protrusions and converts the cell phenotype to round. Flow cyt ometric analysis of the cell shape showed that an inactive analog of Rad bu t not inactive RhoA or inactive cdc42 rounds the cells in the presence of I L-3. Constitutively activated Rad restores the elongated cell phenotype to IL-3-starved cells. We conclude that the activity of Rad is necessary and s ufficient for the IL-3-induced assembly of membrane ruffles. Similar to the IL-3 withdrawal, phorbol 12-myristate 13-acetate (PMA) dissolves actin-for med membrane ruffles and rounds the cells in the presence of IL-3. Flow cyt ometric analysis of the cell shape demonstrated that in the presence of IL- 3 the PMA-induced cell rounding cannot be abolished by constitutively activ e Rad but can be imitated by inactive Rad. These data indicate that PMA dis rupts the IL-3 pathway downstream of Rad. Cells rounded by PMA return to th e elongated phenotype concomitantly with PKC depletion. PMA-induced cell ro unding can be reversed by the PKC-specific inhibitor GF109203X. Experiments with overexpression in Baf3 of individual PKC isoforms and a dominant nega tive PKC-delta indicate that activation of PKC-delta but not other PKC isof orms is responsible for disruption of membrane ruffles. Published 1999 Wile y-Liss, Inc.(dagger)