Gravin (AKAP12, SSeCKS) is a scaffolding protein that acts as a potent inhibitor of tumor metastasis in vivo and in vitro, and regulates morphogenesis during vertebrate gastrulation. Despite being implicated in many cellular processes, surprisingly little is known about the mechanism by which Gravin elicits cell shape changes. In this work, we use in vitro cell spreading assays to demonstrate that the Gravin N-terminus containing the three MARCKS-like basic regions (BRs) is necessary and sufficient to regulate cell shape in vitro. We show that the conserved phosphorylation sites in the BRs are essential for their function in these assays. We further demonstrate that the Gravin BRs are necessary for in vivo function during gastrulation in zebrafish. Together, these results provide an important step forward in understanding the mechanism of Gravin function in cell shape regulation and provide valuable insight into how Gravin acts as a cytoskeletal regulator. (C) 2008 Elsevier Inc. All rights reserved.