Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells

We recently identified hepatoma-derived growth factor (HDGF) as a nuclear t argeted vascular smooth muscle cell (VSM) mitogen that is expressed in deve loping vascular lesions. In the present study, VSM in culture express endog enous HDGF only in the nucleus and target a green fluorescent protein (GFP) -HDGF fusion to the nucleus. To define the features of the HDGF molecule th at are essential for nuclear localization and mitogenic function, deletion and site-directed mutagenesis were performed. Deletion analysis identified the carboxyl-terminal half of HDGF to be responsible for nuclear targeting in VSM. Overexpression of tagged HDGF proteins with point mutations in the putative bipartite nuclear localization sequence in the carboxyl terminus d emonstrated that single Lys --> Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, t he Lys --> Asn mutation of all three lysines blocked nuclear entry. Point m utation of a p34(cdc2) kinase consensus motif within the nuclear localizati on sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple L ys --> Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that H DGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is requi red for HDGF stimulation of DNA replication in VSM.