An encephalitozoon cuniculi ortholog of the RNA polymerase II carboxyl-terminal domain (CTD) serine phosphatase Fcp1.
Fcp1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). The CTD of the microsporidian parasite Encephalitozoon cuniculi consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast. Here we show that E. cuniculi encodes a minimized 411-aa Fcp1-like protein (EcFcp1), which consists of a DxDx(T/V) phosphatase domain and a BRCA1 carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp1 enzymes. Nonetheless, EcFcp1 can function in lieu of Saccharomyces cerevisiae Fcp1 to sustain yeast cell growth. Recombinant EcFcp1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-PO(4)) substrates. EcFcp1 dephosphorylates CTD positions Ser2 and Ser5 with similar efficacy in vitro. We exploit synthetic CTD Ser2-PO(4) and Ser5-PO(4) peptides to define minimized substrates for EcFcp1 and to illuminate the importance of CTD primary structure in Ser2 and Ser5 phosphatase activity.
Hausmann
Stéphane
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Schwer
Beate
B
Shuman
Stewart
S
D000595:Amino Acid Sequence; D000818:Animals; D019313:BRCA1 Protein / Q000737:chemistry; D001665:Binding Sites; D002455:Cell Division; D009670:Encephalitozoon cuniculi / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*
D019313:BRCA1 Protein; D010446:Peptide Fragments; D012694:Serine; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase