In collaboration with the Yee lab, we have recently shown that the transcriptional regulator HBP1 is a new target for the p38 MAP kinase pathway. The p38 MAP kinase signaling pathway participates in both apoptosis and G1 arrest. In contrast to the established role in apoptosis, the documented induction of G1 arrest by activation of the p38 MAP kinase pathway has attracted recent attention with reports of substrates that are linked to cell cycle regulation. Both a p38 MAP kinase docking site (aa 81-125), and a p38 MAP kinase phosphorylation site (serine 401) were identified in the HBP1 protein. Furthermore, the docking and phosphorylation sites on HBP1 were specific for p38 MAP kinase, but not for ERK and JNK family members. In defining the role of p38 MAP kinase regulation, the inhibition of p38 MAP kinase activity was shown to decrease HBP1 protein levels by triggering protein instability, as manifested by a decrease in protein half-life. A mutation of the p38 MAP kinase phosphorylation site at aa 401 ((S-A)₄₀₁HBP1) also triggered HBP1 protein instability. While protein stability was compromised by mutation, the specific activities of (S-A)₄₀₁HBP1) and of wild-type HBP1 appeared comparable for transcriptional repression. Finally, p38 MAP kinase-mediated regulation of the HBP1 protein also contributed to the regulation of G1 progression. These results support a molecular framework in which p38 MAP kinase activity contributes to cell cycle inhibition by stabilizing HBP1 and other G1 regulatory proteins.

Several studies have linked the production of reactive oxygen species (ROS) by the NADPH oxidase to cellular growth control. In many cases, activation of the NADPH oxidase and subsequent ROS generation is required for growth factor signaling and mitogenesis in non-immune cells. We have recently demonstrated that HBP1 regulates the gene for the p47phox regulatory subunit of the NADPH oxidase. The promoter of the p47phox gene contains six tandem high affinity HBP1 DNA binding elements at -1243 to -1318 bp from the transcriptional start site which were required for repression. Furthermore, HBP1 repressed the expression of the endogenous p47phox gene through sequence-specific binding. With HBP1 expression and subsequent reduction in p47phox gene expression, intracellular superoxide production was correspondingly reduced. Using both wild type and a dominant-negative mutant of HBP1, we demonstrated that the repression of superoxide production through the NADPH oxidase contributed to the observed cell cycle inhibition by HBP1. Together, these results indicate that HBP1 may contribute to regulation of NADPH oxidase-dependent superoxide production through transcriptional repression of the p47phox gene. These studies define a transcriptional mechanism for regulating intracellular ROS levels, and has implications in cell cycle regulation.

Current work focuses on the hypothesis that HBP1 represses growth-factor dependent signal transduction through inhibition of growth factor-induced ROS generation, increased PTP activity and a concomitant decrease in ligand-induced signal transduction. The rationale for this focus is two-fold. First, aberrant EGF signaling via EGFR and Her2/neu has a well-defined role in breast cancer progression. Second, screening of human breast cancer samples has shown that HBP1 is mutated in 28% of these cancers, and that these mutated forms of HBP1 are nonfunctional for repression of p47 phox and ROS generation. These clinical HBP1 observations, along with recent data, support our hypothesis that HBP1 may have an important role in EGF-dependent EGFR/Her2/neu function. These pre-clinical investigations, combined with our ongoing clinical HBP1 analysis, should provide a molecular foundation for new biomarkers in the design of specific clinical diagnosis strategies.