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 targeted vascular smooth muscle cell (VSM) mitogen that is expressed in developing vascular lesions. In the present study, VSM in culture express endogenous 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 that 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 demonstrated that single Lys --> Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, the Lys --> Asn mutation of all three lysines blocked nuclear entry. Point mutation of a p34(cdc2) kinase consensus motif within the nuclear localization sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple Lys --> Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that HDGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is required for HDGF stimulation of DNA replication in VSM.