PHOSPHORYLATION REGULATES THE WATER CHANNEL ACTIVITY OF THE SEED-SPECIFIC AQUAPORIN ALPHA-TIP

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family, Expression of alpha-TIP in Xenopus oocytes confered a 4- to 8-fold increase in the osmotic water permeability (P-f) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin, alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated, We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation, First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity, Second, exposure of oocytes to the cAMP agonists and bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min, That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit, Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the P-f of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99, To our knowledge, this is the first evidence of aquaporin regulation via phosphorylation and we propose this process as a mechanism for regulating water permeability of biological membranes.