Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca 2؉ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca 2؉ -ATPase 2 (SERCA2) isoforms, SERCA2a Sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) 1 are single-subunit integral membrane P-type ATPases that mediate the ATP-driven transport of cytosolic Ca 2ϩ against a concentration gradient into the lumen of intracellular Ca 2ϩ -releasable stores such as sarcoplasmic and endoplasmic reticulum (1-4). SERCAs belong to the P-type ATPase family distinguished by the obligatory formation of an aspartyl-phosphorylated intermediate as part of their catalytic cycle. The enzyme cycles reversibly between several states (Scheme 1), of which at least E 1 Ca 2 , E 1 ϳP(Ca 2 ), E 2 -P, and E 2 can be experimentally distinguished. The transfer of the ␥-phosphoryl group of ATP to the aspartate (Asp 351 ) of the phosphorylation domain, leading to the ADP-sensitive high energy phosphoenzyme intermediate E 1 ϳP(Ca 2 ), is activated by conformational changes associated with the binding of two calcium ions in exchange for protons (E 2 to E 1 Ca 2 transition). The conversion of this intermediate to the ADP-insensitive low-energy E 2 -P phosphoenzyme intermediate constitutes a crucial rate-limiting step in Ca 2ϩ translocation (1-3). High resolution models for the atomic structure, generated by x-ray crystallography of crystals in E 1 Ca 2 (5) and Ca 2ϩ -free E 2 (6) forms, and extensive mutational studies (1, 7-11) of the 110-kDa SERCA1a enzyme (994 amino acids) have shown that Ca 2ϩ translocation and ATP utilization are coupled through long range intramolecular interactions between the 10-helix membrane-spanning domain, harboring the Ca 2ϩ binding sites, and the large cytosolic head consisting of actuator (A), phosphorylation (P), and nucleotide-binding (N) domains (Fig. 1).The three human SERCA genes (ATP2A1, ATP2A2, and ATP2A3) encode up to a total of 10 isoforms as a result of the alternative splicing of their pre-mRNA (12-16). Mutations in SERCA genes have been recently detected in human diseases such as Brody disease (muscle disorder) for SERCA1 (17) and
Acrokeratosis verruciformis of Hopf is a localized disorder of keratinization affecting the distal extremities. Onset is early in life and the disease is inherited in an autosomal dominant fashion. Although histology of acrokeratosis verruciformis lesions shows no evidence of dyskeratosis, a possible relationship with Darier's disease has long been postulated on the basis of clinical similarity. ATP2A2 encoding the sarco(endo)plasmic reticulum Ca2+ ATPase2 pump has been identified as the defective gene in Darier's disease. In this report, we studied a family affected with acrokeratosis verruciformis in six generations and identified a heterozygous P602L mutation in ATP2A2. This mutation predicts a nonconservative amino acid substitution in the ATP binding domain of the molecule. The mutation segregates with the disease phenotype in the family and was not found in 50 controls. Moreover, functional analysis of the P602L mutant showed that it has lost its ability to transport Ca2+. This result demonstrates loss of function of the sarco(endo)plasmic reticulum Ca2+ ATPase2 mutant in acrokeratosis verruciformis, thus providing evidence that acrokeratosis verruciformis and Darier's disease are allelic disorders.
Darier's disease is an autosomal dominantly inherited skin disorder characterized by loss of adhesion between epidermal cells, breakdown of desmosome-keratin filaments, and abnormal keratinization. ATP2A2 has been identified as the causative gene of Darier's disease. This gene encodes the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) isoform 2 pump, which transports Ca2+ from the cytosol into the endoplasmic reticulum lumen to maintain a low cytosolic Ca2+ concentration. Using indirect immunofluorescence and biochemical analysis, we investigated the distribution of key desmosomal proteins in normal human and Darier's disease keratinocytes under various calcium conditions. We show that inhibition of SERCA by thapsigargin in normal human keratinocytes impairs the trafficking of the desmoplakins, desmoglein, and desmocollin to the cell surface; these proteins show a diffuse cytoplasmic distribution and, together with plakoglobin, form detergent-insoluble aggregates. In Darier's disease keratinocytes, only the trafficking of desmoplakin is significantly inhibited; in these cells, desmoplakin forms insoluble aggregates when extracted with mild detergent. In contrast, the transmembrane proteins desmoglein and desmocollin are efficiently transported to the cell surface. These proteins, along with plakoglobin, remain equally distributed between detergent-soluble and -insoluble fractions. We also demonstrate an interaction between SERCA2 and desmoplakin during differentiation. Our results provide further insights into the critical role of calcium ATPases in maintaining epidermal integrity.
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