Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells

Abstract

We have examined the effects of depleting lumenal Ca2+ on the synthesis, phosphorylation and secretion of caseins in lactating mouse mammary cells by using inhibitors of the endoplasmic reticulum Ca(2+)-ATPase or the ionophore ionomycin in the absence of external Ca2+. Treatment with these drugs resulted in a transient increase in the cytosolic Ca2+ concentration due to Ca2+ mobilization. Protein synthesis over a 1 h period was substantially inhibited by Ca2+ depletion, but in a pulse-chase protocol secretion of pre-synthesized proteins was unaffected by Ca2+ depletion. Analysis of polysome profiles showed that Ca2+ depletion resulted in a loss of polysomes, consistent with an inhibition of initiation of protein synthesis. Neither treatment with Ca(2+)-ATPase inhibitors to deplete endoplasmic reticulum Ca2+ nor treatment with ionomycin/EGTA had any effect on an early phase of phosphorylation of alpha- or beta/gamma-caseins, but Ca2+ depletion resulted in a decrease in a late phase of casein phosphorylation. These results indicate that lumenal Ca2+ is required to maintain protein synthesis in lactating mammary cells but is not required for protein secretion, and that Ca2+ accumulation in the Golgi cisternae is required for a late but not for an early phase of casein phosphorylation.

Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase

“…Previous work has shown that the putative rat SPCA mRNA and protein are highly expressed in the lactating mammary gland (24,25). Furthermore, the pattern of rat SPCA expression is consistent with the biochemical evidence for a Golgi Ca 2ϩ -ATPase (2,3,7,21,22) in mammary tissue. In this study, we demonstrate that the rat SPCA in located in the Golgi with characteristics of a Ca 2ϩ -ATPase.…”

“…At least two Golgi luminal calcium-binding proteins have been identified that meet the criteria for maintaining the Golgi calcium pool, Cab45 and CALNUC (18,19,27). In mammalian Golgi, biochemical evidence suggests a P-type Ca 2ϩ -ATPase with slightly different characteristics from the plasma membrane Ca 2ϩ -ATPases (PMCA) or SERCAs (2,7,22,32). More recently, the yeast PMR1 gene product was found to be a Ca 2ϩ -ATPase located in the Golgi complex of yeast and Caenorhabditis elegans (28,31).…”

“…Calcium is critical for many cellular functions, including protein processing and secretion by the Golgi (3,7). The Golgi complex contains a large calcium pool in the lactating mammary cell (5), and studies using ion microscopy or electron energy loss imaging analysis have shown that the Golgi complex is a calcium-rich cell compartment in most cells (23).…”

Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment

“…In particular, using a specifically Golgi-targeted aequorin (Go-Aeq) as Ca 2+ sensor, the GA has been demonstrated to release Ca 2+ into the cytoplasm in response to inositol 1,4,5-trisphosphate (IP 3 ) generation and to take up Ca 2+ primarily via the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA), but also, in part, via the secretory pathway Ca 2+ ATPase1 (SPCA1) (2,3,5). On the basis of indirect evidence, it has also been suggested that the Ca 2+ content of the organelle (6) is relevant in the control of several intra-GA processes (7)(8)(9)(10) and indeed mutations of the GA SPCA1 lead to a dominant form of skin pathology, Hailey-Hailey disease (11)(12)(13).…”

Purification of Golgi casein kinase from bovine milk

“…The phosphorylation of caseins is important to allow them to bind Ca# + and for the subsequent formation of casein micelles, which remain stable in milk [3]. The protein kinase responsible for this physiological casein phosphorylation is present within Golgi compartments [4][5][6][7][8], and is only accessible in in itro assays after disruption of the Golgi membranes [6,[9][10][11]. The use of caseins as substrates for protein kinases has resulted in the identification and detailed characterization of two protein kinases : casein kinase (CK) 1 and CK2 [12].…”