Phospho-specific antibodies: more valuable than originally thought.

THE KIDNEY SUCCEEDS IN DETERMINING body composition not only because of the complex arrangement of transporters along the nephron but also because these transporters are regulated by extracellular signals so that the kidney can respond appropriately to a range of physiological conditions. Protein phosphorylation is one of the principal regulatory mechanisms that control the activities and behavior of kinases, channels, enzymes, and structural proteins, so determining their phosphorylation state is an essential part of understanding their regulation. Over the last 20 years, phospho-specific antibodies have been enormously useful in defining regulatory mechanisms that control cell functions. In many laboratories, these reagents are used as definitive assays for the phosphorylation state of proteins, and we forget how difficult life was before their advent. Consequently, the creation of phospho-specific antibodies that recognize two sites in the type 3 Na/H exchanger (NHE3) by Kocinski et al. (4) may not seem like a major achievement. However, these reagents make practical studies that address the fundamental physiological role of NHE3 that would be impossible or extremely cumbersome otherwise. Studies with the first phospho-specific antibody, an antiphosphotyrosine antibody, were published in 1984, and demonstrated phosphorylation of the PDGF receptor in response to binding its ligand, PDGF, in intact cells (1). At that time, antibodies that could immunoprecipitate the PDGF receptor did not exist, and although phosphorylation of the receptor had been demonstrated using purified proteins and standard biochemical techniques, whether this receptor was truly phosphorylated in response to PDGF in vivo could not be determined. The anti-phosphotyrosine antibody was used to immunoprecipitate the PDGF receptor following treatment of the cells with PDGF and P. This antibody allowed partial purification of the receptor to the point that its phosphorylation could be recognized with autoradiography, so that specific phosphorylation of it on tyrosine residues could be demonstrated. This and subsequent anti-phosphotyrosine antibodies led to the specific identification of other proteins containing phosphotyrosines and additional proteins that interact with these proteins. In many cases, these antibodies recognized their targets in cells and tissues, so that the precise location of phosphoproteins in the cell could be determined, analyses that cannot be performed using standard biochemical techniques. Kocinski et al. (4) describe the creation of monoclonal and polyclonal antibodies that recognize two sites on NHE3 that are phosphorylated by PKA, serine 552 and serine 605. NHE3 is one of the principal transporters responsible for Na and HCO3 reabsorption in the kidney and intestine, and consequently, its regulation is essential to allow adaptation to a range of physiological states (7). As would be expected, the activity of this transporter is regulated by multiple mechanisms involving processes that include phosphorylation, dephosphorylation, trafficking, and association with a variety of proteins in response to hormones, changes in osmolarity, blood pressure, and pH (7). Under most experimental conditions, NHE3 is active in its basal state and subject to inhibition by factors that stimulate cAMP and PKA. In response to parathyroid hormone (PTH) and dopamine, NHE3 is phosphorylated on its COOH terminus by PKA at serine 552 and serine 605, its transport activity is decreased, and this decrease in activity is associated with reduced levels of membrane-associated transporters (5, 10). NHE3 interacts with two scaffolding proteins, NHERF and PDZK1. NHERF also binds ezrin, a protein that functions as a PKA-binding protein. PDZK1, a multivalent PDZ domaincontaining protein, also binds D-AKAP-2, another PKA binding protein (2, 3, 6, 8). These scaffolding proteins may enhance the effect of cAMP by localizing the transporter near the kinase and ensuring efficient phosphorylation. Phosphorylation of NHE3 then leads to internalization and reduced activity (9). For practical reasons, these studies of NHE3 regulation have been carried out primarily in cell culture and with expressed proteins. Although the mechanisms identified are almost certainly correct, studies that address the phosphorylation state of NHE3, its interaction with scaffolding proteins, colocalization of kinases, and the way in which these factors determine localization of the transporter in intact tissues and cells have not been performed because current cell biological and biochemical approaches do not permit preservation of tissue and cell structure with simultaneous analysis of the phosphorylation state of NHE3. The paper by Kocinsky et al. (4) in this issue of American Journal of Physiology-Renal Physiology not only describes the production of anti-phosphoserine antibodies that can determine the phosphorylation state of NHE3 at two sites known to be PKA substrates but also demonstrates that under basal conditions, serine 552 is phosphorylated to a greater degree than serine 605 and that the NHE3 that is phosphorylated on serine 552 is preferentially localized to the coated-pit region of the brush border, where it is inactive. These results suggest that the phosphorylation state of NHE3 is an important factor in determining its subcellular localization, and by implication, its pattern of interaction with other proteins. NHE3 may associate with distinct sets of proteins in different cell compartments. The conditions and location within the cell where NHE3 associates with its scaffolding proteins are not fully defined. Although serine 552 and serine 605 are phosphorylated by PKA, they may also be phosphorylated by other kinases, or PKA may be activated under conditions that are independent of classical ligands for receptors that couple to G s and adenylyl cyclase. Segregation of NHE3 in the cell without changes in the phosphorylation state of serine 552 or serine 605 will Address for reprint requests and other correspondence: R. T. Miller, Dept. of Medicine, Case Western Reserve Univ., Div. of Nephrology, Louis Stokes Veterans Affairs Medical Ctr., 10701 East Blvd., Cleveland, OH 44106 (e-mail: [email protected]). Am J Physiol Renal Physiol 289: F247–F248, 2005; doi:10.1152/ajprenal.00077.2005.