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Vol. 12, Issue 1, 185-199, January 2001
Regulatory Subunit But Not SG2NA,
Striatin, or Polyomavirus Middle Tumor Antigen
§
Department of Biochemistry and Winship Cancer Center,
Emory University School of Medicine, Atlanta, Georgia 30322.
Binding of different regulatory subunits and methylation of the
catalytic (C) subunit carboxy-terminal leucine 309 are two important
mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In
this study, both genetic and biochemical approaches were used to
investigate regulation of regulatory subunit binding by C subunit
methylation. Monoclonal antibodies selectively recognizing unmethylated
C subunit were used to quantitate the methylation status of wild-type
and mutant C subunits. Analysis of 13 C subunit mutants showed that
both carboxy-terminal and active site residues are important for
maintaining methylation in vivo. Severe impairment of methylation
invariably led to a dramatic decrease in B
subunit binding but not
of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding.
In fact, most unmethylated C subunit mutants showed enhanced binding to
striatin and SG2NA. Certain carboxy-terminal mutations decreased B
subunit binding without greatly affecting methylation, indicating that
B subunit binding is not required for a high steady-state level of C
subunit methylation. Demethylation of PP2A in cell lysates with
recombinant PP2A methylesterase greatly decreased the amount of C
subunit that could be coimmunoprecipitated via the B subunit but not
the amount that could be coimmunoprecipitated with A subunit or MT.
When C subunit methylation levels were greatly reduced in vivo, B
subunits were found complexed exclusively to methylated C subunits,
whereas striatin and SG2NA in the same cells bound both methylated and
unmethylated C subunits. Thus, C subunit methylation is critical for
assembly of PP2A heterotrimers containing B subunit but not for
formation of heterotrimers containing MT, striatin, or SG2NA. These
findings suggest that methylation may be able to selectively regulate
the association of certain regulatory subunits with the A/C heterodimer.
Portions of this work were performed while these
investigators were in the Division of Cellular and Molecular Biology,
Dana-Farber Cancer Institute, and Department of Pathology, Harvard
Medical School, Boston, MA.
E.O. was supported by an Erwin Schrödinger
Fellowship from Austrian Fonds zur Förderung der
Wissenschaftlichen Forschung. Present address: Institute of Molecular
Biology, Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria.
§
Present address: Oklahoma University Health Science
Center, Oklahoma City, OK 73106.
Corresponding author. E-mail address:
dpallas{at}emory.edu.
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