Calmodulin-dependent protein phosphatase from Neurospora crassa. Molecular cloning and expression of recombinant catalytic subunit.
A cDNA for the catalytic subunit of a calmodulin (CaM)-dependent protein phosphatase was cloned from Neurospora crassa. The open reading frame of 1557 base pairs encoded a protein of Mr approximately 59,580 and was followed by a 3'-untranslated region of 363 base pairs including the poly(A) tail. Based on primer extension analysis, the mRNA transcript in vivo was 2403 base pairs. Expression of this CaM-protein phosphatase mRNA was developmentally regulated, being highest during early mycelial growth; production of the corresponding protein followed mRNA with a time lag of 8-12 h. Polymerase chain reaction amplification of genomic DNA revealed three small introns, the positions of which coincided with those in the mouse gene, indicating evolutionary conservation of these structures. The deduced sequence showed approximately 75% identity with the mammalian homologue, calcineurin, in aligned regions. A region of 40 amino acids preceding the CaM-binding domain was essentially unchanged, suggesting conservation of a crucial interaction site. Three small segments in the carboxyl half of the protein were unrelated to the mammalian gene and may constitute "variable regions" that confer substrate specificity to the enzyme. An active recombinant catalytic subunit was expressed in bacteria and purified by CaM-Sepharose chromatography. This preparation was stimulated 2- 3-fold by CaM and showed a p-nitrophenol phosphatase activity equal to that of the bovine brain holoenzyme, although its dephosphorylation of phosphoprotein substrates was markedly different. These findings demonstrate that the catalytic subunit of this phosphatase can exhibit high activity in the absence of its intrinsic Ca(2+)-binding subunit.
Higuchi
S
S
Section on Immunology, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852.
Tamura
J
J
Giri
P R
PR
Polli
J W
JW
Kincaid
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D009492:Neurospora crassa; D016366:Open Reading Frames; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D002148:Calmodulin-Binding Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases