Ras signals for the transformation of mammalian cells are apparently transduced through Rho GTPases. The Rho GTPase family member Cdc42 generates independent signals that regulate the rearrangement of the actin cytoskeleton and the transcription of genes. However, the molecular mechanism of signal transduction from Cdc42 to the nucleus remains to be understood. The nonreceptor tyrosine kinases ACK-1 and ACK-2 have been found to bind specifically to Cdc42. In this paper we studied whether ACKs transduce Cdc42 signals to the nucleus directly, or through other cytoplasmic proteins. Using immunocytochemistry and Western blot analysis, we found a nuclear localization of ACKs in semi-confluent glioblastoma (U251) cells, as opposed to a cytosolic localization in confluent cells. In agreement with the nuclear localization, a putative nuclear export signal was identified in ACK-1 and ACK-2. Furthermore, the interaction of Cdc42 with ACKs was shown to be essential for the nuclear localization of ACKs. Overexpression of ACK42 (a Cdc42 binding domain of ACK) inhibited cell growth and movement, indicating that Cdc42 signals are transduced to the nucleus through ACKs. This is the first report providing evidence of a novel role for ACKs in transducing Cdc42 signals directly to the nucleus. (C) 2003 Elsevier Inc. All rights reserved.