The role of protein phosphorylation in renal amino acid transport.
Changes in tubular reabsorption of amino acids and other solutes are characteristic of the immature renal tubule and of various hereditary nephropathies. The cellular mechanisms governing these aberrations in renal amino acid transport have not been established. Calcium (Ca2+)-dependent protein kinases are known to phosphorylate membrane-bound carrier proteins, thereby modulating transport of various solutes by the proximal tubule. The role of these enzymes in regulating renal tubular amino acid transport, particularly during kidney development, is unknown. We investigated: (1) the effect of Ca(2+)- and phospholipid-dependent protein kinase [protein kinase C (PKC)] and Ca2+/calmodulin-dependent protein kinase II (CaMKII) on sodium chloride (NaCl)-linked proline transport by renal brush border membrane vesicles (BBMV) from adult rats using the "hypoosmotic shock" technique (lysis of vesicles); (2) the activity, expression and subcellular distribution (cytosol, particulate, BBM) of Ca(2+)-dependent protein kinases in kidneys from 7-day-old and adult rats using MBP 4-14 and autocamtide II phosphorylation assays for PKC and CaMKII, respectively, endogenous protein phosphorylation (using gel electrophoresis and autoradiography) and Western immunoblot analysis to detect PKC and CaMKII. The studies showed: (1) endogenous (membrane-bound) CaMKII and PKC as well as exogenous, highly purified PKC inhibit proline uptake by phosphorylated, lyzed/resealed BBMV when compared with control vesicles; the voltage-clamped, nonelectrogenic component of proline transport was inhibited by PKC- but not CaMKII-mediated phosphorylation; (2) a Ca(2+)-dependent activity of both kinases was evident in all subcellular fractions tested in immature and adult kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)
Zelikovic
I
I
Department of Pediatrics, University of Washington, Seattle.
Przekwas
J
J
D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D007684:Kidney Tubules / Q000196:embryology / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism
D011392:Proline; D012964:Sodium; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium