REGULATING MITF'S ACTIVATION OF OSTEOCLAST TARGET GENES

描述(申请人提供)：小眼炎相关转录因子(MITF)和TFE3是基本的螺旋-环-亮氨酸拉链转录因子，在破骨细胞中表达。我们已经证明了MITF和TFE3可以激活破骨细胞分化的重要基因。MITF和TFE3表达缺失的小鼠是成骨细胞，这证明了MITF和TFE3是破骨细胞分化所必需的证据。通过酵母双杂交筛选，我们鉴定了CDC25C相关蛋白激酶1(C-TAK1)和蛋白酶体盖子的成分POH1与MITF的氨基末端相互作用。这一建议试图1)确定MITF在破骨细胞中是如何转移到细胞核的，2)确定影响破骨细胞中MITF稳定性的蛋白质。为了确定C-TAK1和MITF之间的相互作用(目标1)，我们将确定MITF的哪个氨基酸残基被C-TAK1磷酸化，确定哪些MITF氨基酸残基是与14-3-3蛋白质结合的关键，并确定C-TAK1和14-3-3相互作用对MITF细胞定位的影响。14-3-3是一个蛋白质家族，识别特定的磷酸化丝氨酸，并结合和隔离细胞质中的蛋白质。为了确定MITF和POH1的S相互作用的影响(目的2)，我们将确定MITF的稳定性是否受到脑脊液-1和RANKL信号的影响，定位MITF与POH1相互作用的区域，并确定POH1‘S与MITF相互作用对破骨细胞靶基因MITF激活的影响。通过了解MITF如何激活成骨细胞特异性基因，药物靶点可能被认为是控制破骨细胞分化和激活的治疗方法。

DESCRIPTION (provided by applicant): Microphthalmia-associated transcription factor (Mitf) and Tfe3 are basic helix-loop-helix leucine zipper transcription factors that are expressed in osteoclasts. We have demonstrated that Mitf and Tfe3 colloborate to activate genes important for osteoclast differentiation. Evidence that Mitf and Tfe3 are necessary for osteoclast differentiation is demonstrated by the fact that mice null for expression of Mitf and Tfe3 are osteopetrotic. Using a yeast two hybrid screen, we identified Cdc25C-associated kinase 1 (C-TAK1) and POH1, a component of the proteasome lid, as interacting with the amino terminus of Mitf. This proposal attempts to 1) determine how Mitf is translocated to the nucleus in osteoclasts and 2) to identify proteins that affect Mitfs stability in osteoclasts. To define the interaction between C-TAK1 and Mitf (aim 1), we will determine which amino acid residue of Mitf is phosphorylated by C-TAK1, determine which Mitf amino acid residues are critical for binding to 14-3-3 proteins and determine what effect C-TAK1 and 14-3-3 interaction has on Mitfs cellular location. 14-3-3 are a family of proteins that recognize specific phosphorylated serines and bind to and sequester a protein in the cytoplasm. To determine the effect of Mitf and POH1's interaction (aim 2), we will determine if Mitf's stability is affected by CSF-1 and RANKL signaling, map the region of Mitf that interacts with POH1 and determine the effect of POH1's interaction with Mitf on Mitfs activation of osteoclast target genes. By understanding how Mitf activates ostoeclast specific genes, drug targets may be suggested that will lead to therapies to control the differentiation and activation of osteoclasts.