Abstract
In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase alpha-subunit and PLMS showed the presence of alpha and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E1 approximately P --> E2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E2(K) --> E1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase alpha-subunit is important for the modulation of shark Na,K-ATPase activity.
Highlights
In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS
Both the E1ϳP 3 E2-P reaction, which is partially rate-limiting in shark, and the K؉ deocclusion reaction, E2(K) 3 E1, are accelerated. The latter may explain the finding that the apparent Na؉ affinity was increased by the specific C-terminal PLMS truncation. These data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase ␣-subunit is important for the modulation of shark Na,K-ATPase activity
Because the 3Ј-end of the amplicon exhibited no homology to any known FXYD gene, specific sense (3Ј-RACE, 1; 3Ј-RACE, 2) and antisense (5Ј-RACE, 1; 5Ј-RACE, 2) primers were synthesized 5Ј to this region where there was still nucleotide and amino acid homology to other FXYD proteins
Summary
In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be cleaved Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. The latter may explain the finding that the apparent Na؉ affinity was increased by the specific C-terminal PLMS truncation These data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase ␣-subunit is important for the modulation of shark Na,K-ATPase activity. Such interactions are especially well described for the regulation of SERCA1 by phospholamban (PLN) and sarcolipin [3–7]
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