Note: Descriptions are shown in the official language in which they were submitted.
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METHYLATION OF ARGININE AND RELATED AMINO ACIDS
FIELD OF THE INVENTION
This invention relates generally to the measurement
of the methylation of arginine and related amino acids, and
more particularly to the methylation of SLM-1, SLM-2, Sam68,
hnRNP K and WASP in particular.
BACKGROUND OF THE INVENTION
Proteins that provide an intracellular link from the
extracellular milieu are called signal transduction proteins.
These proteins contain numerous post-translation modifications
including tyrosine phosphorylation (Pawson, 1995) and arginine
methylation (Gary and Clarke, 1998). The tyrosine kinases and
the arginine methyltransferases that modify the signal proteins
are often found associated with growth factor receptors and are
involved in transferring the information to the inside of the
cell. It is known that many growth factor receptors and
soluble tyrosine kinases are oncogenes and can transform cells.
Thus substrates of tyrosine kinases or post-translational
modifications in response to growth factor receptors can serve
as an indication of whether or not a cell is cancerous. In
addition to being post-translationally modified, many proteins
contain proline motifs and phosphotyrosine residues that serve
as specific binding sites for SH3 and SH2 domain containing
proteins (Pawson, 1995).
Post-translation modification includes N-methylation
of the side chain of the guanidine arginine residues. Related
amino acids, such as lysine and histidine, can also be subject
of methylation.
Three mammalian protein arginine methyltransferases
have been cloned including PRMT1 (Scott et al., 1998 and Lin
et al., 1996), PRMT2 (Scott et al., 1998) and PRMT3 (Tang
et al., 1998). From the EST database, it has been predicted
that others will be identified (Tang et al., 1998). Arginine
methylation is thought to be an irreversible post-translational
modification. Protein arginine N-methyltransferase enzymes
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catalyze the formation of asymmetric 1V~,IV~-dimethylarginine
residues in proteins by transferring methyl groups from
S-adenosylmethionine to the guanidino nitrogen atoms of
arginine. These enzymes generally methylate arginine in the
context of RGG or arginine-glycine rich domains.
Known substrates for PRMTs include hnRNP proteins
including hnRNP A1 and myelin basic protein. PRMT1 interacts
with several proteins including the cytoplasmic domain of the
interferon alpha receptor 1 (Abramovich et al, 1997) and the
immediate early gene products including TIS21 and BTG1 interact
with PRMT1 (Lin et al., 1996). These findings suggest that
PRMT1 activity may be regulated by cell surface receptors and
may be involved in signal transduction aspects of downstream of
the interferons. PRMT1 is predominantly nuclear and methylates
arginines that contain glycines at their C-terminus in repeats
such as arginine-glycine or arginine-glycine-glycine and so
forth (Scott et al., 1998 and Lin et al., 1996). PRMT2 has not
been shown to have any arginine methyltransferase activity,
perhaps because the appropriate substrate has not been
identified (Scott et al., 1998). Interestingly, PRMT2 contains
an SH3 domain in its sequence and thus has the potential to
link arginine methyltransferase to signalling proteins. It is
well known that SH3 domains bind proline rich repeats that are
frequently found in cytoskeletal and signalling proteins
(Pawson, 1995). PRMT3 is predominantly cytoplasmic and also
favours the methylation of arginine amino acids that contain a
C-terminal glycine (Tang et al., 1998).
SUMMARY OF THE INVENTION
The subject invention provides for methods of
assaying methylation of arginine and related amino acids, more
particularly methods of assaying arginine methyltransferase
activity by means of measuring the methylation of suitable
substrates, such as SLM-1, SLM-2, Sam68, hnRNP K or WASP
proteins. The invention is described with respect to the
specific methyltransferase substrates SLM-1, SLM-2, Sam68,
hnRNP K and WASP, however, it will be appreciated that other
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substrates can be methylated in a similar fashion, and
accordingly the invention has application to such substrates
although it is described herein with reference to SLM-1, SLM-2,
Sam68, hnRNP K and WASP.
The subject invention also provides for a method of
measuring the percentage of methylated substrate relative to
total substrate in a cell. The methylation of substrate such
as SLM-1, SLM-2, Sam68, hnRNP K or WASP may be used to
determine whether or not a cell is transformed or has the
potential to become cancerous. The methylation of WASP may
also be used to diagnose individuals with Wiskott-Aldrich
syndrome. Another aspect of the invention is to provide for
antibodies that may be used to detect polypeptides that contain
methylated arginines.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
This invention relates to the measurement of
methylation of arginine and related amino acids such as lysine
and histidine. The invention is described with respect to
arginine, however, it will be appreciated that related amino
acids, such as lysine and histidine, can be methylated in a
similar fashion, and accordingly the invention has similar
application to lysine and histidine although it is described
herein with reference to arginine.
By arginine methylation or by methylated arginines,
it is meant that arginine residues in a peptide contain one or
more methyl group. By methylation, it is meant
1V~-monomethlyarginine (MMA) , N~,1V~-dimethylarginine residues
(asymmetric, DMA) and N~,N'~-dimethylarginine (symmetric, DMA').
Antibodies specific for arginine methylation would be
able to recognize MMA, DMA and/or DMA' in a polypeptide and/or
oligopeptide.
Those skilled in the art will appreciate that there
must exist a protein module that binds methylated arginine
residues much like SH2 domains bind phosphotyrosine residues.
As hereinbefore mentioned the invention relates to a
method for assaying a medium for the presence of a substance
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that affects a methylarginine specific antibody or
methylarginine binding domain to a methylarginine ligand
regulatory system comprising a specific methylarginine binding
antibody or protein module or a subdomain thereof, and a
methylated ligand which is capable of interacting with said
specific methylarginine binding antibody or protein module or a
subdomain thereof to form a specific methylarginine binding
antibody or protein module- methylated ligand complex, said
specific methylarginine binding antibody or protein module or
subdomain and/or said methylated ligand being present in a
known concentration, and incubating with a substance which is
suspected of affecting a specific methylarginine binding
antibody or protein module-methylated ligand regulatory system,
under conditions which permit the formation of said specific
methylarginine binding antibody or protein module methylated
ligand complex, and assaying for said specific methylarginine
binding antibody or protein module methylated ligand complex,
free specific methylarginine binding antibody or protein module
or subdomains thereof, or non-complexed methylated ligand.
Purified derivatives of SLM-1, SLM-2, Sam68, hnRNP K and WASP
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their
derivatives may be purified from a variety of cells. By
"purified" it is meant, when referring to a peptide or
nucleotide sequence, that the indicated molecule is present in
the substantial absence of other biological macromolecules,
e.g., polypeptides, polynucleic acids, and the like of the same
type. The term "purified" as used herein preferably means at
least 95% by weight, more preferably at least 99.8% by weight,
of biological macromolecules of the same type present (but
water, buffers, and other small molecules, especially molecules
having a molecular weight of less than 1000 can be present).
The term "pure" as used herein preferably has the same
numerical limits as "purified" immediately above. The term
"isolated" as used herein refers to polypeptide or
polynucleotide molecules separated not only from other
peptides, or from DNAs or RNAs, respectively, that are present
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in the natural source of the macromolecule but also from other
macromolecules and preferably refers to a macromolecule found
in the presence of (if anything) only a solvent, buffer, ion or
other component normally present in a solution of the same.
5 "Isolated" and "purified" do not encompass either natural
materials in their native state or natural materials that have
been separated into components (e.g., in an acrylamide gel) but
not obtained either as pure substances or as solutions.
Suitable cell sources for the production of purified
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives
include cells naturally producing SLM-1, SLM-2, Sam68, hnRNP K,
or WASP including most mammalian cells, cells not naturally
encoding an expressible SLM-1, SLM-2, Sam68, hnRNP K, or WASP
gene but genetically modified to do so, and cells naturally
producing SLM-1, SLM-2 and Sam68 but genetically modified so as
to produce elevated levels of SLM-1, SLM-2, Sam68, hnRNP K, or
WASP. Preferred cell sources for SLM-1, SLM-2, Sam68, hnRNP K,
WASP and their derivative molecules are selected so that at
least 5%, preferably at least 50% and more preferably at least
90% of the SLM-1, SLM-2, Sam68, hnRNP K, WASP or their
derivative molecules are methylated. Purification methods for
SLM-l, SLM-2, Sam68, hnRNP K, WASP and their derivatives that
depend on affinity reagents specific for methylated arginines,
necessarily employ SLM-1, SLM-2, Sam68, hnRNP K, WASP and their
derivatives isolated from cells that methylate SLM-1, SLM-2,
Sam68, hnRNP K, WASP and their derivatives that have been
produced in cells that lack arginine methyltransferase activity
but have been methylated in vitro with enzymes with arginine
methyltransferase activity.
It will be appreciated that an important advantage of
the subject invention is to apply recombinant DNA techniques so
as to provide for cellular lysates that contain SLM-1, SLM-2,
Sam68, hnRNP K, or WASP in significantly higher (at least
2-fold, and preferably at least 10-fold) concentrations than
found in naturally occurring cells or cell lines that have not
been modified by exogenous SLM-1, SLM-2, or Sam68 encoding
nucleic acid sequences. Since SLM-1, SLM-2, Sam68, hnRNP K,
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and WASP derivatives are not naturally produced, it is apparent
that cells from which SLM-1, SLM-2, Sam68, hnRNP K, and WASP
derivatives can be isolated do not naturally encode SLM-1,
SLM-2, Sam68, hnRNP K, or WASP derivatives but are genetically
modified to do so.
Cells from which SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives may be isolated include both prokaryotic
and eukaryotic cells. Preferred cellular sources for the
isolation of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their
derivatives include mammalian cells possessing high levels of
arginine methyltransferase activity. Of particular interest
are mammalian cells transfected with arginine
methyltransferases such as PRMT1, PRMT2, and PRMT3. Other
mammalian cell sources of interest for the purification of
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives
include mammalian cells stimulated by growth factors that bind
to growth factor receptors that stimulate arginine
methyltransferase activity. Another preferred source for
preparations from which to purify SLM-1, SLM-2, Sam68, hnRNP K,
WASP and their derivatives is insect cells, preferably grown in
tissue culture, and genetically modified by baculovirus
expression vectors or the like to express SLM-1, SLM-2, Sam68,
hnRNP K, WASP and their derivatives and an arginine
methyltransferase. A particularly preferred source of SLM-1,
SLM-2, Sam68, hnRNP K, or WASP derivatives is the SF9 cell
line. Another source would be to produce recombinant SLM-1,
SLM-2, Sam68, hnRNP K, or WASP in bacteria or yeast as a fusion
protein with tags such as histidine repeats or glutathione
S-transferase proteins. It will be appreciated that purified
SLM-1, SLM-2, Sam68, hnRNP K, or WASP can be methylated
in vitro to yield purified methylated SLM-1, SLM-2, Sam68,
knRNP K, or WASP.
Purification of SLM-1, SLM-2, Sam68, hnRNP K, or WASP
Derivatives
Affinity purification of SLM-1, SLM-2, Sam68,
hnRNP K, or WASP and their derivatives may employ various
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immobilized reagents specific for SLM-1, SLM-2, Sam68, hnRNP K,
WASP or their derivatives, i.e., affinity reagents. The
affinity purification may be performed in batches or employ
chromatography columns. The affinity reagents may be
immobilized to a variety of inert matrices prepared in bead
form. Suitable immobilization matrices include cross-linked
agarose beads, Sepharose, cross-linked polyacrylamide beads,
Sephacryl, and the like. When the affinity reagents used are
antibodies, a preferred immobilizing matrix is protein A
sepharose. Affinity reagents of interest include antibodies,
purified SH3 domains, purified SH2 domains, and homopolymeric
RNA. Purification of methylated SLM-1, SLM-2, Sam68, hnRNP K,
or WASP can be performed by using methylated arginine specific
antibodies. Purification of non-methylated SLM-1, SLM-2,
Sam68, hnRNP K, WASP or their derivatives may also be achieved
through the use of SLM-l, SLM-2, Sam68, hnRNP K, or WASP
specific antibodies as affinity reagents.
In addition to production of purified SLM-1, SLM-2,
Sam68, hnRNP K, WASP and their derivatives by purification of
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives
produced in cells, purified SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives may be produced by organic chemical
reactions performed in vitro. Automated equipment for the
direct synthesis of the polypeptides disclosed herein is
commercially available. Such equipment provides convenient
access to peptides of the invention, either by direct synthesis
or by synthesis of a series of fragments that can be coupled
using other known techniques. The use of such commercially
available polypeptide synthesis machines and the like are a
preferred method of synthesizing oligopeptides SLM-1, SLM-2,
Sam68, hnRNP K or WASP derivatives having about 5-25 amino
acids. It will be appreciated by those skilled in the art that
oligopeptides can be synthesized containing arginines that are
methylated.
Other methods for synthesis of SLM-1, SLM-2, Sam68,
hnRNP K, WASP and their derivatives include the in vitro
transcription of DNA sequences encoding SLM-1, SLM-2, Sam68,
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hnRNP K, WASP and their derivatives coupled with the in vitro
translation of the RNA transcripts thus produced. In vitro
transcription systems are well known in the art. In vitro
transcription systems typically involve the creation of
nucleotide sequences in which the coding sequence of interest
is located downstream from a strong promoter, such as a
promoter specific for SP-6 or T7 RNA polymerases, followed by
the addition of an RNA polymerase specific for the promoter,
and substrates required for the reaction. Similarly, in vitro
translation systems are well known in the art and may be used
to produce SLM-1, SLM-2, Sam68, hnRNP K, WASP and their
derivative polypeptides from a variety of transcripts produced
by in vitro transcription systems.
Uses for methylarginine specific antibodies
The invention provides for antibodies capable of
recognizing polypeptides containing methylated arginines. By
the term "antibodies," it is intended both polyclonal and
monoclonal antibodies with natural immunoglobulin sequences,
synthetic antibody derivatives, and the like; antibodies may
be modified so as to be joined to any of a variety of labels,
fluorescent, radioactive, enzymatic, biotin/avidin or the like.
Synthetic antibody derivatives include natural immunoglobulin
sequences that have been mutated and selected for altered
binding specificity, various immunoglobulin gene derived
polypeptides, typically single chain, produced by genetically
modified bacteria, antibodies modified so as to contain
modified constant regions and the like; a review of such
synthetic antibody derivatives based on the principles of
antibody formation is provided in Harlow and Lane, Antibodies,
Coldspring Harbor Laboratory, Coldspring Harbor Press, A
Laboratory Manual, 1988.
Antibodies specific for methylated arginines may be
generated by using proteins or peptides that contain one or
more methylated arginines. By induction of antibodies it is
intended not only the stimulation of an immune response by
injection into animals, but analogous steps in the production
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of synthetic antibodies such as the screening of recombinant
immunoglobulin libraries (Orlandi et al., 1989 or Huse et al.,
1989), or the in vitro stimulation of lymphocyte populations.
Of particular interest is the development of antibody
preparations, monoclonal antibodies, specific for methylated
arginine containing polypeptides, i.e., monospecific
antibodies.
Short oligopeptides, i.e., containing about 20 amino
acids or less, are of particular interest for both the
induction and the screening of mono-specific antibodies
specific for epitopes of interest. In general, oligopeptides
for use in the induction of epitope specific monospecific
antibodies will have an amino acid sequence corresponding to at
least a portion of the epitope of interest.
It is also of interest to produce antibody
preparations that are capable of specifically binding to the
methylated forms of SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Uses for such methylation state detecting antibodies include
the measurement of the degree of methylation of SLM-1, SLM-2,
Sam68, hnRNP K, or WASP in a cell.
Assays
The subject invention provides methods and reagents
for performing assays capable of measuring the amount of
arginine methyltransferase activity present in a cell and the
fraction of proteins that contain methylated arginines.
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their
derivatives may be used as substrates for the detection and
quantification of arginine methyltransferase activity from a
variety of cellular sources. It is desirable to measure
arginine methyltransferase activity for several reasons. Of
particular interest is the measurement of arginine
methyltransferase activity produced by arginine
methyltransferase encoded by oncogenes and proto-oncogenes.
Thus assays for arginine methyltransferase may be employed to
determine whether a cell is cancerous or has cancer potential.
Also of interest is the measurement of arginine
CA 02266760 1999-04-08
methyltransferase activity attributable to the stimulation
membrane bound ligand receptors associated with arginine
methyltransferase activity, since the extent of methylation of
SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be used to measure
5 the extent to which ligands are binding to receptors.
Arainine methyltransferase assays
Arginine methyltransferase assays of interest measure
the rate of methylation of SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives by arginine methyltransferase in a cell,
10 rather than simply measuring the amount of methylated SLM-l,
SLM-2, Sam68, hnRNP K, or WASP present in a cell. Thus
arginine methyltransferase assays of interest employ a method
for distinguishing arginine methylation events that take place
during an assay from arginine methylation events that occur
before an assay. Arginine methyltransferase assays may employ
the step of adding a methyl group, preferably S-adenosyl
methionine and the like, to an assay mixture containing
suitable buffers and salts. Methyl sources may be
radioactively labelled on the terminal carbon or hydrogens so
as to provide for the detection of arginine methyltransferase
activity.
Arginine methyltransferase assays employing
radioactive labels may or may not employ the step of addition
of SLM-l, SLM-2, Sam68, hnRNP K, WASP or their derivatives,
because arginine methyltransferase substrates initially present
in the cell, or SLM-1, SLM-2, Sam68, hnRNP K, WASP or their
derivatives added externally, and subsequently methylated by a
radioactive methyl source, may subsequently be isolated by
addition of SLM-1, SLM-2, Sam68, hnRNP K, or WASP-specific
antibodies, followed by the step of radiometric quantitation.
Generally it will be preferable to add SLM-1, SLM-2, Sam68,
hnRNP K, or WASP, preferably produced by recombinant means, to
the assay mixture. After the arginine methyltransferase
reaction has been allowed to progress, the amount of
radioactive label incorporated into SLM-1, SLM-2, Sam68,
hnRNP K, or WASP is measured by radiometric means. In order to
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measure the amount of labelling, the unincorporated label must
be removed prior to radiometric measurement. This removal can
be achieved through a variety of means including
immunoprecipitation of SLM-1, SLM-2, Sam68, hnRNP K or WASP
with anti-SLM-1, SLM-2, Sam68, hnRNP K or WASP antibodies.
An important advantage of the subject invention is
that the polypeptides provided for permit the detection and
quantification of arginine methyltransferase activity without
requiring the addition of radioactively labelled methyl groups.
Methods for measuring arginine methyltransferase activity
without the addition of radioactively labelled methyl groups
include assays involving the use of (1) SLM-1, SLM-2, Sam68,
hnRNP K or WASP derivatives that contain epitopes not present
on SLM-1, SLM-2, Sam68, hnRNP K, or WASP, (2) antibodies
specific for that epitope, and (3) anti-methylated-arginine
antibodies or protein domains that specifically bind to
arginine methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Such assays involve the addition of the SLM-l, SLM-2, Sam68,
hnRNP K, or WASP derivatives to the assay mixture, followed by
the immunoprecipitation or immunobilization of the SLM-1,
SLM-2, Sam68, hnRNP K, or WASP derivative by means of the
epitope specific antibody so as to separate the SLM-1, SLM-2,
Sam68, hnRNP K or WASP derivative from other cellular proteins
containing methylated arginines (including endogenous SLM-1,
SLM-2, Sam68, hnRNP K, and WASP).
In addition to providing methods and reagents for use
in the detection of arginine methyltransferase activity present
in a cell, the subject invention provides methods and reagents
for determining what fraction of the SLM-1, SLM-2, Sam68,
hnRNP K, or WASP in a cell is methylated as well as determining
the absolute amount of methylated SLM-1, SLM-2, Sam68, hnRNP K,
or WASP present in a cell. By cell, it is intended not only
individual cells, but multiple cells. Arginine methylation of
SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be detected by a
variety of means. If the methyl donor in the assay contains a
radioactive label, then arginine methyltransferase activity may
be detected by separating the labelled SLM-1, SLM-2, Sam68,
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hnRNP K, or WASP from the unincorporated label and quantitating
the amount of label incorporated into the SLM-l, SLM-2, Sam68,
hnRNP K, or WASP substrate. When non-radioactively labelled
methyl donors are used in assays, methylated SLM-1, SLM-2,
Sam68, hnRNP K, or WASP may be detected by means of generally
known immunoassays in which the immunospecific reagent employed
is specific for methylated arginines.
The subject invention provides for methods and
reagents for performing assays capable of determining what
fraction of SLM-1, SLM-2, Sam68, hnRNP K, or WASP in a cell is
methylated. Such assay may employ well known immunoassay
technology such as ELISA, RIA, western blotting, and the like.
The use of SLM-1, SLM-2, Sam68, hnRNP K, or WASP specific
antibodies (as well as SLM-1, SLM-2, Sam68, hnRNP K, WASP and
their derivatives) as provided for by the subject invention may
be used in connection with the preciously described well-
established immunoassay technology, in order to provide for
assays capable of detecting the extent of SLM-1, SLM-2, Sam68,
hnRNP K, or WASP methylation in a cell. In general, such
assays will employ two types of SLM-1, SLM-2, Sam68, hnRNP K,
or WASP specific antibodies (or similar binding reagent) in an
immobilized phase, namely (1) antibodies capable of binding
SLM-l, SLM-2, Sam68, hnRNP K, or WASP in both methylated and
non-methylated form or antibodies capable of binding only the
non-methylated form of SLM-1, SLM-2, Sam68, hnRNP K, or WASP,
and (2) antibodies capable of binding the methylated SLM-1,
SLM-2, Sam68, hnRNP K, or WASP. By employing two types of
specific binding reagent, it is possible to determine the
relative quantities of the methylated and unmethylated forms of
SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in a sample. The
binding of SLM-1, SLM-2, Sam68, hnRNP K, or WASP (methylated
and non-methylated) to an immobilized antibody phase may be
detected by the addition of a third antibody, preferably
labelled, and having an affinity for exposed epitopes on the
antibody bound SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Comparison of binding of the labelled antibody to SLM-1, SLM-2,
Sam68, hnRNP K, or WASP bound to the 2 different types of
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immobilized antibody may be used to determine the fraction of
methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP present among
the total SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in the
sample.
The invention having been described, the following
examples are offered to illustrate the subject invention by way
of illustration, not by way of limitation.
Example 1
Identifying protein arginine methyltransferase substrates.
HeLa cells (0.5 x 106) were transfected with 5 ug of
each plasmid expressing myc epitope tagged mouse SLM-1
(Di Fruscio et al., 1999), mouse SLM-2 (Di Fruscio et al.,
1999), mouse Sam68 (Wong et al., 1992), brine shrimp GRP33
(Cruz-Alvarez and Pellicer, 1987), human hnRNP K (Michael
et al., 1997) and mouse Qkl (Ebersole et al., 1996) as
described previously (Richard et al., 1995). Twelve hours
after transfection the cells were lysed in 500 ul lysis buffer
(1% Triton X-100, 20 mM Tris-HC1 pH 7.4, 150 mM NaCl, 50 mM
NaF, 100 uM sodium vanadate, 0.01% phenylmethanesulfonyl
fluoride, 1 ug/ml aprotinin, and 1 ug/ml leupeptin) on ice for
15 minutes. The debris was removed by centrifugation and the
supernatant was divided equally into two fractions. Half was
immunoprecipitated with control immunoglobulin G and the other
half was immunoprecipitated with the anti-myc antibody 9E10
(American Tissue Culture Collection) for 1 hour on ice. Then
20 ul of 50% slurry of protein A Sepharose was added and
incubated with end-over-end mixing for 30 minutes. The
immunoprecipitated proteins were washed extensively and 2 ug of
purified recombinant GST-PRMT1 and 2 ul of
[3H-methyl]-adenosyl-1-methionine was added in 25 mM Tris-HCl
pH 7.4, 1 mM EDTA for 30 minutes at room temperature. PRMT1 is
a protein arginine methyltransferase that uses the methyl
groups from S-adenosyl-methionine to transfer them to the
guandino nitrogens of arginine (Gary and Clarke, 1998). The
reaction was stopped by adding 20 ul of 2X Laemmli buffer
(Laemmli, 1970) and loaded on an SDS 10%-polyacrylamide gel.
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The gel was then soaked in 3H Enhance (ICN) for 30 minutes and
then the gel was equilibrated with water for 1 hour, dried and
exposed to film. It was observed that in myc
immunoprecipitates Sam68, SLM-1, SLM-2, GRP33, and hnRNP K but
not Qk1 were labelled with PRMT1. These data demonstrate that
SLM-1, SLM-2, Sam68, GRP33 and hnRNP K are substrates for
methyltransferase in vitro and may also be substrates for
methyltransferase in vivo. Type I methyltransferases are known
to methylate arginines that contain a C-terminal glycine (Gary
and Clarke, 1998), and indeed the methylated proteins contain
numerous RG repeats.
Example 2
Preparation of antibodies which recognize dimethylarainine
residues.
Peptides were synthesized that correspond to the
arginine-glycine repeats of Sam68, hnRNP K and Wiskott-Aldrich
syndrome protein (WASP). These peptides were synthesized
unmethylated or with the arginines containing asymmetric
dimethylarginines. The peptides for Sam68 were:
Sam68 P3 (GVSVRGRGAAPPPPPVPRGRGVGP)
Sam68 P3* (GVSVR*GR*GAAPPPPPVPR*GR*GVGP)
wherein R* denotes dimethylarginine
Sam68 P4 (TRGATVTRGVPPPPTVRGAPTPR)
Sam68 P4* (TR*GATVTR*GVPPPPTVR*GAPTPR)
wherein R* denotes dimethylarginine
P3* and P4* were coupled by the amino terminus to KLH and
injected in rabbits to generate rabbit polyclonal antibodies.
The goal of generating these antibodies is to have antibodies
that would recognize all dimethylated arginines in polypeptides
or at the very least recognize the dimethylated arginines in
the context of the peptide generated. Thus, in the former
situation the antibody may recognize any protein or peptide
that contains dimethylated arginines. In the latter situation,
the antibody would recognize only dimethylated Sam68 and no
other protein. After analyzing the antisera obtained from the
P3* injected rabbit and the P4* injected rabbit both situations
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were obtained. The anti-sera from the rabbit injected with P4*
only recognized P4* and had some cross-reactivity to the
unmethylated Sam68 P4 peptides. This antibody did not
recognize P3 methylated or unmethylated. The anti-sera from
5 the rabbit injected with P3*, interestingly, recognized many
peptides that contained dimethylated arginines. This anti-sera
did not recognize the unmethylated counterpart of these
peptides. These analyses were performed by using the ELISA.
Other peptides that were used for these analyses include:
10 Sam68 PO (RLTPSRPSPLPHRPRGGGGGPRGG)
Sam68 PO* (RLTPSRPSPLPHRPR*GGGGGPR*GG)
wherein R* denotes dimethylarginine
WASP P1 biotin-RQEPLPPPPPPSRGGNQLPR
WASP P1* biotin-RQEPLPPPPPPSR(*)GGNQLPR
15 wherein R* denotes dimethylarginine
WASP P2 biotin-APPPPTPRGPPPPGRGGPPPPPP
WASP P2* biotin-APPPPTPR(*)GPPPPGR(*)GGPPPPPP
wherein R* denotes dimethylarginine
hnRNPK P1 biotin-SPRRGPPPPPPGRGGRGGSR
hnRNPK P1* biotin-SPRR(*)GPPPPPPGR(*)GGR(*)GGSR
wherein R* denotes dimethylarginine
hnRNPK P2 biotin-RARNLPLPPPPPPRGGDL
hnRNPK P2* biotin-RARNLPLPPPPPPR(*)GGDL
wherein R* denotes dimethylarginine
These studies demonstrate that antibodies can be generated that
will recognize dimethylarginine residues in polypeptides. The
presence of dimethylated arginines in a medium can be monitored
and examined by using the medium to disrupt the interaction
between a peptide containing dimethylated arginines and a
methylarginine specific antibody. As such it is possible to
examine whether a given medium contains polypeptides with
dimethylated arginines.
Example 3
Effect of methylation on binding domains.
The Sam68 P3 peptide binds the SH3 domains of p59f~"'
and PLC~y-1 (Richard et al., 1995). HeLa cells were lysed and
CA 02266760 1999-04-08
16
incubated with the SH3 domains of p59fs'I' and PLCy-1 covalently
coupled to beads in the absence or presence of unmethylated or
methylated peptides. It was observed that P3 or P4 peptides
described in Example 2 competed out the binding of Sam68 to the
SH3 domains, but that the methylated version of those peptides
P3* or P4* could not compete out the binding of Sam68 to the
SH3 domain of p59f~ and PLCy-1. These data suggest that
methylated peptides cannot bind certain SH3 domain such as
those of p59f}"' and PLCy-1. Thus, these data suggest that most
likely a novel protein module exists that has the ability to
bind dimethylated arginine residues, much like phosphotyrosine
polypeptides associate with SH2 and PTB domain containing
proteins (Pawson, 1995).
CA 02266760 1999-04-08
17
References
Abramovich et al, 1997 EMBO J. 16:260-266
Cruz-Alvarez, M. and Pellicer, A. 1987. Cloning of a full-
length complementary cDNA for as Artemia salina glycine-rich
protein. J. Biol. Chem., 262:13377-13380.
Di Fruscio, M., Chen, T. and Richard, S. 1999. Two novel Sam68-
like mammalian proteins SLM-1 and SLM-2: SLM-1 is a Src
substrate during mitosis, Proc. Natl. Acad. Sci. USA, 96:2710-
2715.
Ebersole, T.A., Chen, Q., Justice, M.J. and Artzt, K. 1996. The
quaking gene unites signal transduction and RNA binding in the
developing nervous system. Nature Genetics, 12:260-265.
Gary, J.D. and Clarke, S. 1998. RNA and protein interactions
modulated by protein arginine methylation. Prog. Nuc. Acid Res.
Mol. Biol. 61:65-131.
Huse et al., 1989 Science 256: 1275-1281
Laemmli, U.K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 227:680-685.
Lin et al., 1996 JBC 271:15034-44
Michael W. M., Eder, P.S. and Dreyfuss, G. 1997. The K nuclear
shuttling domain: a novel signal for nuclear import and nuclear
export in the hnRNP K protein. EMBO J., 16:3587-3598.
Orlandi et al., 1989 PNAS USA 86:3833-3837
Pawson, T. 1995. Protein modules and signalling networks.
Nature, 373:573-580.
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Richard, S. Yu, D., Blumer, K.J., Hausladen, D., Olszowy, M.W.,
Connelly, P.A. and Shaw, A. S. 1995. Association of p62, a
multi-functional SH2- and SH3-binding protein, with src-family
tyrosine Kinases, Grb2, and phospholipase Cg-1. Mol. Cell.
Biol., 15:186-197.
Scott et al., 1998 Genomics 48:330-340
Tang et al., 1998 JBC 273: 16935-45
Wong, G., Muller, O., Clark, R., Conroy, L., Moran, M.F.,
Polakis, P. and McCormick, F. 1992. Molecular cloning and
nucleic acid binding properties of the GAP-associated tyrosine
phosphoprotein p62. Cell, 69:551-558.
CA 02266760 1999-04-08
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Sequences
Figure 1. Amino acid sequence of SLM-1 - ID NO: 1.
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library
(A) ORGANISM: Mus musculus
MGEEKYLPELMAEKDSLDPSFVHASRLLAEEIEKFQGSDGKKEDEEKKYL-50
DVISNKNIKLSERVLIPVKQYPKFNFVGKLLGPRGNSLKRLQEETGAKMS-100
ILGKGSMRDKTKEEELRKSGEAKYAHLSDELHVLIEVFAPPGEAYSRMSH-150
ALEEIKKFLVPDYNDEIRQEQLRELSYLNGSEESGRGRGIRGRGIRITPT-200
APSRGRGGAVPPPPPPGRGVLTPRGTTVTRGALPVPPIARGVPTPRARGT-250
AAVPGYRAPPPPAHDAYEEYGYDDGYGGEYDDQTYEAYDNSYVTPTQSVP-300
EYYDYGHGVNEDAYDSYAPEEWATTRSSLKAPPPRSARGGYREHPYGRY-349
Figure 2. Amino acid sequence of SLM-2 - ID N0: 2.
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library
(A) ORGANISM: Mus musculus
MEEKYLPELMAEKDSLDPSFTHALRLVNREIEKFQKGEGKEEEKYIDWI-50
NKNMKLGQKVLIPVKQFPKFNFVGKLLGPRGNSLKRLQEETLTKMSILGK-100
CA 02266760 1999-04-08
GSMRDKAKEEELRKSGEAKYFHLNDDLHVLIEVFAPPAEAYARMGHALEE-150
IKKFLIPDYNDEIRQAQLQELTYLNGGSENADVPWRGKSTLRTRGVTTP-200
AITRGRGGVTARPVAVGVPRGTPTPRGVLSTRGPVSRGRGLLTPRARGVP-250
PTGYRPPPPPPTQETYGEYDYDDGYGTAYDEQSYDSYDNSYSTPAQSAAD-300
5 YYDYGHGLSEDAYDSYGQEEWTNSRHKAPSARTAKGVYRDQPYGRY-346
Figure 3. Amino acid sequence of Sam68 - ID NO: 3.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 443 amino acids
10 (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: full-length ORF
15 (vi) ORIGINAL SOURCE: brain library
(A) ORGANISM: Mus musculus
MQRRDDPASRLTRSSGRSCSKDPSGAHPSVRLTPSRPSPLPHRPRGGGGG-50
PRGGARASPATQPPPLLPPSTPGPDATWGSAPTPLLPPSATAAVKMEPE-100
NKYPPELMAEKDSLDPSFTHAMQLLSVEIEKIQKGESKKDDEENYLDLFS-150
20 HKNMKLKERVLIPVKQYPKFNFVGKILGPQGNTIKRLQEETGAKISVLGK-200
GSMRDKAKEEELRKGGDPKYAHLNMDLHVFIEVFGPPCEAYALMAHAMEE-250
VKKFLVPDMMDDICQEQFLELSYLNGVPEPSRGRGVSVRGRGAAPPPPPV-300
PRGRGVGPPRGALVRGTPVRGSITRGATVTRGVPPPPTVRGAPTPRARTA-350
GIQRIPLPPTPAPETYEDYGYDDTYAEQSYEGYEGYYSQSQGESEYYDYG-400
HGELQDSYEAYGQDDWNGTRPSLKAPPARPVKGAYREHPYGRY-443
Figure 4. Amino acid sequence of hnRNPK - ID NO: 4.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
CA 02266760 1999-04-08
21
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library
(A) ORGANISM: human
METEQPEETFPNTETNGEFGKRPAEDMEEEQAFKRSRNTDEMVELRILLQ-50
SKNAGAVIGKGGKNIKALRTDYNASVSVPDSSGPERILSISADIETIGEI-100
LKKIIPTLEEGLQLPSPTATSQLPLESDAVECLNYQHYKGSDFDCELRLL-150
IHQSLAGGIIGVKGAKIKELRENTQTTIKLFQECCPHSTDRWLIGGKPD-200
RWECIKIILDLISESPIKGRAQPYDPNFYDETYDYGGFTMMFDDRRGRP-250
VGFPMRGRGGFDRMPPGRGGRPMPPSRRDYDDMSPRRGPPPPPPGRGGRG-300
GSRARLPLPPPPPPRGGDLMAYDRRGRPGDRYDGMVGFSADETWDSAIDT-350
WSPSEWQMAYEPQGGSGYDYSYAGGRGSYGDLGGPIITTQVTIPKDLAGS-400
IIGKGGQRIKQIRHESGASIKIDEPLEGSEDRIITITGTQDQIQNAQYLL-450
QNSVKQYSGKFF-462
Figure 5. Amino acid sequence of WASP - ID NO: 5.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 520 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library
(A) ORGANISM: mouse
MNSGPGPVGGRPGGRGGPAVQQNIPSNLLQDHENQRLFELLGRKCWTLAT-50
TWQLYLALPPGAEHWTMEHCGAVCFVKDNPQKSYFIRLYGLQAGRLLWE-100
QELYSQLVYLTPTPFFHTFAGDDCQVGLNFADESEAQAFRALVQEKIQKR-150
NQRQSGERRQLPPPPAPINEERRGGLPPVPPHPGGDHGGPSGGPLSLGLV-200
TVDIQNPDITSSRYRGLPAPGPGPTDKKRSGKKKISKADIGAPSGFKHVS-250
HVGWDPQNGFDVNNLDPDLRSLFSRAGISEAQLTDAETSKLIYDFIEDQG-300
GLEAVRQEMRRQEPLPPPPPPCRGGGGGGGGGGGGGGGGGGQPLRPPWG-350
SNKGRSGPLPPVPMGGAPPPPTPRGPPPPGRGGPPPPPPPATGRSGPPPP-400
CA 02266760 1999-04-08
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PLPGAGGPPAPPPPPPPPPPPPCPGSGPAPPPLPPTPVSGGSPAPGGGRG-450
ALLDQIRQGIQLNKTPGALENSVQQPPAQQSEGLVGALMHVMQKRSRVIH-500
SSDEGEDQTGEDEEDDEWDD-520
Abbreviations for the amino acid
residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly;
H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln;
R, Arg; S,Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
