THE ROLE OF PROTEIN-PHOSPHORYLATION IN RENAL AMINO-ACID-TRANSPORT

Changes in tubular reabsorption of amino acids and other solutes are c haracteristic of the immature renal tubule and of various hereditary n ephropathies. The cellular mechanisms governing these aberrations in r enal amino acid transport have not been established. Calcium (Ca2+)-de pendent protein kinases are known to phosphorylate membrane-bound carr ier proteins, thereby modulating transport of various solutes by the p roximal tubule. The role of these enzymes in regulating renal tubular amino acid transport, particularly during kidney development, is unkno wn. We investigated: (1) the effect of Ca2+- and phospholipid-dependen t protein kinase [protein kinase C (PKC)] and Ca2+/calmodulin-dependen t protein kinase II (CaMKII) on sodium chloride (NaCl)-linked proline transport by renal brush border membrane vesicles (BBMV) from adult ra ts using the ''hypoosmotic shock'' technique (lysis of vesicles); (2) the activity, expression and subcellular distribution (cytosol, partic ulate, BBM) of Ca2+-dependent protein kinases in kidneys from 7-day-ol d and adult rats using MBP 4-14 and autocamtide II phosphorylation ass ays for PKC and CaMKII, respectively, endogenous protein phosphorylati on (using gel electrophoresis and autoradiography) and Western immunob lot analysis to detect PKC and CaMKII. The studies showed: (1) endogen ous (membrane-bound) CaMKII and PKC as well as exogenous, highly purif ied PKC inhibit proline uptake by phosphorylated, lyzed/resealed BBMV when compared with control vesicles; the volltage-clamped, nonelectrog enic component of proline transport was inhibited by PKC- but not CaMK II-mediated phosphorylation; (2) a Ca2+-dependent activity of both kin ases was evident in all subcellular fractions tested in immature and a dult kidneys. An increased activity of PKC and CaMKII in the cytosol a nd BBM was evident in immature kidneys when compared with adult kidney s. Autoradiographic analysis revealed a Ca2+-dependent protein kinase- mediated phosphorylation of several low and high molecular weight prot eins in kidneys from both age groups. Immunoblot analysis detected alp ha and beta isoenzymes of PKC in the cytosol and BBM of kidneys from b oth ages, CaMKII alpha in the BBM of both age groups and CaMKII beta i n the cytosol of adult kidneys. These data suggest that Ca2+-dependent protein kinase-mediated phosphorylation inhibits NaCl-dependent proli ne transport across the luminal membrane. While CaMKII-induced inhibit ion of proline transport is secondary to a kinase-mediated modulation of electrolyte flux, PKC-mediated inhibition is in part due to a direc t phosphorylation of the proline carrier. The protein kinase-induced i nhibition of amino acid transport is similar to the inhibitory effect of these enzymes on other Na+-dependent transport mechanisms in the pr oximal tubule. Age-related changes in the activity of Ca2+-dependent p rotein kinases and differential regulation of their isoenzymes during kidney development may play an important role in renal cell growth and differentiation and in the developmental maturation of renal tubular transport.