Protein phosphorylation in Golgi, endosomal, and endoplasmic reticulum membrane fractions of lacrimal gland

Abstract

Ca²⁺/calmodulin-and cAMP-dependent protein kinase activities were characterized in two subcellular membrane samples. Membranes from rat lacrimal gland were isolated by differential and density gradient centrifugation into six density windows. The present study focused on membranes from density windows III and V which contain mixtures of apical, Golgi, endosomal, and endoplasmic reticulum membranes in different proportions. Phosphorylation of membrane proteins was measured by incubating the samples in [g-³²P]ATP and separating the proteins by discontinuous SDS-PAGE followed by autoradiography. The amount of phosphate incorporated into specific peptide bands was quantified by densitometry. Ca²⁺/calmodulin-dependent protein kinase phosphorylated a 52,000 MW peptide in membranes from both density windows with a maximal increase from 0.3 to 66 μM free Ca²⁺. Trifluoperazine and promethazine, two inhibitors of Ca²⁺/calmodulin-dependent protein kinases, inhibited this phosphorylation. cAMP-dependent protein kinase phosphorylated a 22,000 MW peptide and a 91,000 MW peptide which were present in membranes from density window III only. We conclude that a Ca²⁺/calmodulin-dependent protein kinase activity is present in membranes from both density window III and V whereas a cAMP-dependent protein kinase activity is present only in membranes from density window III.

Key Words:

• calmodulin
• cAMP
• lacrimal gland
• phosphorylation
• protein kinases