Protein phosphatase 1 catalytic subunit isoforms from alfalfa: biochemical characterization and cDNA cloning.
The catalytic subunit of protein phosphatase 1 (PP1c) was purified from an alfalfa (Medicago sativa) microcallus cell culture. The preparation was inhibited by rabbit muscle inhibitor-2 and okadaic acid and had a molecular mass of 35 kDa. Five distinct cDNAs termed MsPP1alpha, -beta, -gamma, -delta, and -epsilon were cloned from a M. sativa somatic embryo library. MsPP1alpha was identical to a cDNA reported earlier [A. Páy, M. Pirck, L. Bögre, H. Hirt, and E. Heberle-Bors Mol. Gen. Genet. 244, 176-182, 1994], while the others represented novel isoforms encoded by separate genes. The predicted amino acid sequences of MsPP1alpha, -beta, -gamma, -delta, and -epsilon were highly similar to each other and to other known PP1c sequences. The GST-MsPP1ss fusion protein expressed in Escherichia coli was catalytically active and was inhibited by inhibitor-2 and okadaic acid. Affinity-purified polyclonal MsPP1antipeptide antibody detected a protein of 36 kDa in crude cell extracts. These results proved that the cDNA clone encoded an active PP1c which was very similar to the purified enzyme. The mRNA and protein concentrations of PP1c as well as the specific activity of protein phosphatase 1 did not change during the cell cycle in a synchronized alfalfa cell culture. On the other hand, the isoforms exhibited different steady-state mRNA levels in different plant organs suggesting tissue-specific functions.
Vissi
E
E
Department of Medical Chemistry, University Medical School of Debrecen, Debrecen, Hungary.
Tóth
E C
EC
Kovács
I
I
Magyar
Z
Z
Horváth
G V
GV
Bagossi
P
P
Gergely
P
P
Dudits
D
D
Dombrádi
V
V
D000595:Amino Acid Sequence; D000906:Antibodies; D016590:Aphidicolin / Q000494:pharmacology; D015152:Blotting, Northern; D015139:Blotting, Southern; D020134:Catalytic Domain; D002453:Cell Cycle / Q000187:drug effects; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D015870:Gene Expression / Q000187:drug effects; D015723:Gene Library; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000455:Medicago sativa / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018514:Plant Structures / Q000235:genetics; D054645:Protein Phosphatase 1; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D016590:Aphidicolin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1