Calmodulin‐dependent protein phosphorylation and calcium uptake in rat‐liver microsomes

Abstract

A 20-kDa protein becomes phosphorylated in a process stimulated by Ca2+ and calmodulin in the light microsomal fraction of rat liver homogenate. The uptake of Ca2+ in light microsomal fraction is also calmodulinstimulated. The stimulation of Ca2 + transport is associated with the operation of a protein phosphorylation system dependent on Ca2+ and calmodulin. Transport is inhibited by a protein phosphatase which is stimulated by Ca2+ and calmodulin. It is proposed that the phosphorylation of the 20-kDa protein, which is stimulated by Ca2+ and calmodulin, plays a role in the regulation of the microsomal Ca2+, Mg2+-ATPase. [6]). The third is the mitochondrial transport system, which consists of the electrophoretic uptake pathway and of a Na+/Ca2+ (or H+/CaZ+) exchange system which mediates Ca2 + release (for a recent review see [7]). It is generally assumed that microsomes play an important role in the regulation of the Ca2 + level in liver, and may be involved in some types of toxininduced liver injury [S], as well as in some hormonal signalresponse cascade [9].In a previous paper from this laboratory it has been demonstrated [lo] that the postmitochondrial supernatant of rat liver contains two vesicular fractions which transport Ca2+ actively. The heavier fraction is enriched in plasma membrane markers, and contains a Ca2+-pumping ATPase and a Na+/Ca2 + exchanger. The light microsomal fraction does not possess Na+/CaZ+ exchange activity, and its ATP-driven Ca2 + uptake is relatively insensitive to vanadate. The Ca2 + uptake in this fraction is activated by a cytosolic fraction and by CAMP, and inhibited by the CAMP-dependent protein kinase inhibitor. The anticalmodulin drug trifluoperazine inhibits Ca2 + uptake and Ca2 +-dependent protein phosphorylation in liver microsomes [lo]. This finding has focused our attention on the possible involvement of calmodulin and a protein kinase in the regulation of the Ca2+, Mg2+-ATPase in these membranes.The results presented here show that indeed protein phosphorylation and Ca2+ uptake in rat liver microsomes are regulated by calmodulin. They also show that the latter process is regulated by a Ca2 +-dependent protein phosphorylationdephosphorylation cycle.While the manuscript was in preparation, a report by Moore and Kraus-Friedman has appeared [IOa] on the purification of the liver microsomal Ca2+, Mg2+-ATPase by calmodulin affi ni t y chromatography . ~~Abbreviations. CaM, calmodulin; N,-['251]CaM, azidoiodocalmodulin ; PAGE, polyacrylamide gel electrophoresis ; SDS, sodium dodecyl sulphate. MATERIALS AND METHODS Membrane preparationAlbino male Wistar-strain rats weighing 120 -150 g were killed by decapitation and the livers immediately removed and cut into small pieces in 0.25 M sucrose buffered with 5 mM Tris/Cl pH 7.4, and containing 1 mM mercaptoethanol and 0.5 mM phenylmethylsulfonyl fluoride (buffer A). One liver was homogenized in 70 ml of this medium using a glass Potter homogenizer with two passages of a loosely fitting teflon pestle. The homogenate was filtered t...

Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland

“…Interestingly, chlorpromazine at 100pM inhibited both Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake. The latter effect is consistent with the purported regulation of ATPdependent Ca2+ transport by calmodulin in various cell types (Moore & Kraus-Friedman, 1983;Famulski & Carafoli, 1984). However, it is unknown whether the inhibition of Na+/Ca2+ exchange by chlorpromazine in parotid basolateral membranes also involves a calmodulin-dependent process, as has been reported for cardiac sarcolemma (Caroni & Carafoli, 1983).…”

Visualization of the Ca 2+ -transport system of the mitotic apparatus of sea urchin eggs with a monoclonal antibody

“…Since microtubules have been shown to be Ca2+ sensitive (34), and calmodulin is known to occur in the spindle (35)(36)(37), this hypothesis seems to be applicable to the mitotic apparatus. The range of Ca2' concentrations, to which the vesicles are sensitive, is comparable to that of systems requiring calmodulin (38)(39)(40)(41). The isolated Ca2+-uptake vesicles are susceptible to all known calmodulin inhibitors even though they do not respond to calmodulin (42).…”

Calcineurin

“…The phosphorylation by CAMP-dependent protein kinase and the dephosphorylation by calcineurin of polypeptides associated with the calcium channel (Hosey et al, 1986) supports the hypothesis that phosphorylation modulates the activity of the voltage-dependent Ca2+ channel (Chad and Eckert, 1986). The Ca2+ transport of sarcoplasmic reticulum is known to be regulated by CAMP and Ca2+ and calmodulin-regulated protein kinases (as reviewed by Tada et al, 1978); the phosphatase involved has not yet been isolated but Famulski and Carafoli (1984) reported the inhibitory effect of calcineurin on Ca2+ transport by liver microsomes. None of these effects has been studied extensively and definitive conclusions cannot be drawn but the subject is worthy of further work.…”