Identification of protein kinases involved in the modulation of the dual-affinity nitrate transporter CHL1

• Main Authors: Chih-Hsuan Chuang, 莊智軒
• Other Authors: Yi-Fang Tsay
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/03611508544319613979

碩士 === 國立陽明大學 === 遺傳學研究所 === 93 === CHL1, a nitrate transporter in Arabidopsis was shown to have dual-affinity nitrate uptake activity. Switching between the two modes of CHL1 is regulated by phosphorylation at threonine residue 101. Previous studies indicated that CHL1 can be phosphorylated in vitro by PKA (cAMP-dependent protein kinase). In Arabidopsis genome, there is a putative PKA gene At2g20040. High-affinity nitrate uptake is normal in the T-DNA inserted mutant of At2g20040 suggesting that either At2g20040 is not responsible for CHL1 phosphorylation, or T-DNA insertion (near the 3’ end of the gene) did not disrupt the function of this protein. Moreover, to our surprise, 5’RACE and RT-PCR analysis indicated that At2g20050, At2g20040 together with the intergenic region between At2g20040 and At2g20050 is transcribed encoding a protein with ~120kD in size. This is a novel protein with phosphatase in the N-terminal, and kinase in the C-terminal, the two cAMP/cGMP binding sites between the phosphatase and kinase domains. In-gel protein kinase assay was performed to identify the protein kinases recognize and phosphorylate CHL1 peptide containing T101. Three protein kinase signals with sizes of p110、p60 and p45 were found. Compared with signals found in no peptide sybstrate control, p45 could be autophosphorylated, p110 and p60 may be responsible for CHL1 phosphorylation. Finally, in-gel protein kinase assay was also performed to identify the protein kinases activated by nitrate. According to the results of one-dimensional electrophoresis, we found most the activity of protein kinases activated by nitrate reached climax after 15-minute nitrate treatment. Further, using two-dimensional electrophoresis analysis we found 7 signals after 15-minute nitrate treatment. But, 2 signals was also found in no nitrate treatment control.