ACTIN-BIDING PROTEIN-280 BINDS THE STRESS-ACTIVATED PROTEIN-KINASE (SAPK) ACTIVATOR SEK-1 AND IS REQUIRED FOR TUMOR-NECROSIS-FACTOR-ALPHA ACTIVATION OF SAPK IN MELANOMA-CELLS

SEK-1, a dual specificity protein kinase that serves as one of the imm ediate upstream activators of the stress-activated protein kinases (SA PKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segmen t of ABP-280, upstream of the ABP carboxyl-terminal dimerization domai n. Activation of SEK-1 in situ increases the SEK-1 activity bound to A BP-280 without changing the amount of SEK-1 polypeptide bound. The inf luence of ABP-280 on SAPK regulation was evaluated in human melanoma c ells that lack ABP-280 expression, and in stable transformants of thes e cells expressing wild type ABP, or an actin-binding but dimerization -deficient mutant ABP (ABP Delta CT109). ABP-280-deficient cells show an activation of SAPK in response to most stimuli that is comparable t o that seen in ABP-280-replete cells; ABP-280-deficient cells, however , fail to show the brisk tumor necrosis factor-alpha (TNE-alpha) activ ation of SAPK seen in ABP-replete cells and have an 80% reduction in S APK activation by lysophosphatidic acid. Expression of the dimerizatio n-deficient mutant ABP-280 fails to correct the defective SAPK respons e to lysophosphatidic acid, but essentially normalizes the TNF-alpha a ctivation of SAPK. Thus, a lack of ABP-280 in melanoma cells causes a defect in the regulation of SAPK that is selective for TNF-alpha and i s attributable to the lack of ABP-280 polypeptide itself rather than t o the disordered actin cytoskeleton that results therefrom. ABP-280 pa rticipates in TNF-alpha signal transduction to SAPKs, in part through the binding of SEK-1.