Methylation of the C-terminal leucine residue (Leu309) of protein serine/threonine phosphatase 2A catalytic subunit (PP2AC) is known to regulate catalytic activity in vitro, but the functional consequence(s) of this post-translational modification in the context of the cell remain unclear. Alkali-induced demethylation of PP2AC in purified PP2A heterotrimer (ABαC), but not in purified PP2A heterodimer (AC), indicated that a larger fraction of PP2AC is carboxymethylated in ABαC than in AC. To explore the role of Leu309 in PP2A holoenzyme assembly, epitope-tagged PP2A catalytic subunit (HA-PP2A) and a mutant of HA-PP2A containing an alanine residue in place of Leu309 (HA-PP2A-L309A) were transiently expressed in COS cells. Both recombinant proteins exhibited serine/threonine phosphatase activity when immunoisolated from COS cell extracts. HA-PP2A, but not HA-PP2A-L309A, was carboxymethylated in vitro. A chromatographic analysis of cell extracts indicated that most endogenous PP2AC and HA-PP2A were co-eluted with the A and Bα regulatory subunits of PP2A, whereas most HA-PP2A-L309A seemed to elute with the A subunit as a smaller complex or, alternatively, as free catalytic (C) subunit. The A subunit co-immunoisolated with both tagged proteins; however, substantially less Bα subunit co-immunoisolated with HA-PP2A-L309A than with HA-PP2A. These results demonstrate that the reversibly methylated C-terminal leucine residue of PP2AC is important for Bα regulatory subunit binding. Furthermore, the results provide evidence for an interrelationship between PP2AC carboxymethylation and PP2A holoenzyme assembly.
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Research Article|
April 08 1999
Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Bα subunit
Jeffrey C. BRYANT;
Jeffrey C. BRYANT
1Department of Pharmacology, Room 424 MRB1, Vanderbilt University Medical Center, Nashville, TN 37232-6600, U.S.A.
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Ryan S. WESTPHAL;
Ryan S. WESTPHAL
1Department of Pharmacology, Room 424 MRB1, Vanderbilt University Medical Center, Nashville, TN 37232-6600, U.S.A.
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Brian E. WADZINSKI
Brian E. WADZINSKI
1
1Department of Pharmacology, Room 424 MRB1, Vanderbilt University Medical Center, Nashville, TN 37232-6600, U.S.A.
1To whom correspondence should be addressed (e-mail brian.wadzinski@mcmail.vanderbilt.edu).
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Publisher: Portland Press Ltd
Received:
September 07 1998
Revision Received:
January 13 1999
Accepted:
February 08 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 339 (2): 241–246.
Article history
Received:
September 07 1998
Revision Received:
January 13 1999
Accepted:
February 08 1999
Citation
Jeffrey C. BRYANT, Ryan S. WESTPHAL, Brian E. WADZINSKI; Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Bα subunit. Biochem J 15 April 1999; 339 (2): 241–246. doi: https://doi.org/10.1042/bj3390241
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