Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron 1. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron 1 is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes.

Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G 1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr 85 immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr 85 to Ala 85 resulted in nuclear accumulation of PTHrP, while mutation to Glu 85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation-and cell cycle-dependent, and such control further supports a potential intracellular role (10, 34, 35) for PTHrP.Parathyroid hormone-related protein (PTHrP) 1 is widely expressed (1-3) and acts as a paracrine, and possibly an autocrine and intracrine, factor. However, its intracellular roles have not been fully defined. Recently a nucleolar localization signal (NLS) was identified within PTHrP and deletion of this motif prevented PTHrP from entering the nucleolus, maintaining it as a cytoplasmic protein (4). Nucleolar localization of PTHrP delays apoptosis in chondrocytes (4) and increases smooth muscle cell proliferation (5). PTHrP has also been linked to the ras signaling pathway (6) and the hedgehog signaling pathways (7,8), indicating its importance in regulating growth and differentiation. PTHrP expression is cell cycle-dependent (9, 10) and PTHrP mRNA expression responds to mitogenic factors only at the G 1 phase of the cell cycle (10). Furthermore, PTHrP localizes to the nucleolus at the G 1 phase of the cell cycle (10).Cyclin-dependent kinases control the progression of the various phases of the cell cycle (11). CDKs are activated at different phases of the cell cycle by the formation of cyclin-CDK complex and deactivated when their cyclin partner is degraded. The prototype CDK is CDC2, which associates with cyclin B and regulates the transition between the G 2 and M phases of the cell cycle. Cyclin E-CDK2 and cyclin A-CDK2 complexes are involved in the G 1 to S transition, while CDK4 and CDK6 associating with the D-type cyclins are involved in the progression through G 1 (12). In addition to direct regulation of the cell cycle, cyclins and CDKs have functions in other biological processes such as transcriptional control (13), and protein phosphorylation by CDC2 and CDK2 results in increase of affinity for the cytoplasm of some molecules containing an NLS (14).A stretch of basic residues, or a pair of basic residues separated by a 10 -12 amino acid spacer to form a bipartite NLS, characterizes NLSs. The archetypal protein used in nuclear localization studies is the SV40 T-a...

MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

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