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The p38 mitogen-activated protein (MAP) kinase signaling pathway participates in both apoptosis and G[sub 1] arrest. In contrast to the established role in apoptosis, the documented induction of G[sub 1] arrest by activation of the p38 MAP kinase pathway has attracted recent attention with reports of substrates that are linked to cell cycle regulation. Here, we identify the high-mobility group box protein HBP1 transcriptional repressor as a new substrate for p38 MAP kinase. Our previous work had shown that HBP1 inhibits G[sub 1] progression in cell and animal models, and thus indicated that HBP1 could be a relevant substrate for p38 MAP kinase in cell cycle regulation. In the present work, a p38 MAP kinase docking site (amino acids [aa] 81 to 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. 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. Consistently, a decrease in protein levels was accompanied by a decrease in overall DNA binding activity. A mutation of the p38 MAP kinase phosphorylation site at aa 401 [(S-A)[sub 401]HBP1] also triggered HBP1 protein instability. While protein stability was compromised by mutation, the specific activities of (S-A)[sub 401]HBP1 and of wild-type HBP1 appeared comparable for transcriptional repression. This comparison of transcription-specific activity highlighted that p38 MAP kinase regulated HBP1 protein levels but not the intrinsic activity for DNA binding or for transcriptional repression. Finally, p38 MAP kinase-mediated regulation of the HBP1 protein also contributed to the regulation of G[sub 1] progression. Together, our work supports a molecular framework in which p38 MAP kinase activity contributes to cell cycle inhibition by increasing HBP1 and other G[sub 1] inhibitory factors by regulating protein stability. [ABSTRACT FROM AUTHOR]