(Received for publication, August 3, 1994; and in revised form, December 2, 1994)
From the
We have recently identified a new member of the Ras/GTPase
superfamily termed Rad which has unique sequence features and is
overexpressed in the skeletal muscle of humans with type II diabetes
(Reynet, C., and Kahn, C. R.(1993) Science, 262,
1441-1444). When expressed in bacteria as a glutathione S-transferase fusion protein, Rad bound
[-
P]GTP quickly and saturably. Binding was
specific for guanine nucleotides and displayed unique magnesium
dependence such that both GTP and GDP binding were optimal at
relatively high Mg
concentrations (1-10
mM). Rad had low intrinsic GTPase activity which was greatly
enhanced by a GTPase-activating protein (GAP) activity present in
various tissues and cell lines. Several known GAPs had no stimulatory
effect toward Rad. Conversion of Ser to Asn at position 66 in Rad
(equivalent to position 12 in Ras) resulted in a total loss of GTP
binding. Mutation of Pro
(equivalent to Gly
in Ras) or Gln
(equivalent to Gln
in
Ras) had no effect on Rad GTPase activity, whereas creation of a double
mutation at these positions resulted in exceptionally high intrinsic
GTPase activity. In vitro, Rad was phosphorylated by the
catalytic subunit of cAMP-dependent protein kinase (PK). Phosphopeptide
mapping indicated two PKA phosphorylation sites near the
C
terminus. Rad also co-precipitated a serine/threonine
kinase activity from extracts of various tissues and cell lines which
catalyzed phosphorylation on Rad but was not inhibited by PKA
inhibitor. Thus, Rad is a GTP-binding protein and a GTPase which has
some structure/function similarities to Ras, but displays unique
features. Rad may also be phosphorylated on serine/threonine residues
by PKA and other kinases, as well as regulated by its own GAP which is
present in many tissues and cell types.
The Ras superfamily of GTP-binding proteins has been implicated in a wide spectrum of cellular functions, including cell proliferation and differentiation(1) , intracellular vesicular trafficking(2) , and cytoskeletal control(3) . Since the initial observation that microinjection of a neutralizing Ras antibody into Xenopus oocytes blocked insulin-stimulated maturation(4) , evidence for the involvement of Ras-related proteins in the actions of insulin and other growth factors has accumulated at an increasingly rapid pace(5, 6, 7, 8, 9) . Two members of Ras-related family of proteins, Rab3D (10) and Rab4 (11) , have been implicated in the translocation of glucose transporters in response to insulin, and Rac has been implicated in insulin-stimulated membrane ruffling(12) . By screening cDNA subtraction libraries, our laboratory has identified a transcript encoding a novel member of the Ras superfamily(13) . This protein has unique sequence features compared to other Ras-like proteins and is overexpressed in skeletal muscle of humans with Type II diabetes as compared with non-diabetic and Type I diabetic humans. We termed this protein Rad for Ras-related protein associated with diabetes. Very recently, a second member of the Rad family, termed Gem, was identified by polymerase chain reaction display following activation of T-lymphocytes(14) .
Rad and Gem have several
unique structural features among the Ras/GTPase superfamily which might
affect GTP binding or GTPase activity. Both Rad and Gem are longer than
Ras due to NH- and COOH-terminal extensions; both lack the
CAAX box at the COOH terminus which may affect their ability
to undergo posttranslational modifications, such as
geranyl-geranylation or farnesylation(15) ; and both contain a
number of nonconserved sequence changes in regions G1, G2, and G3 which
are known to be involved in GTP binding and GTP hydrolysis (see Fig. 1). (
)
Figure 1: Comparison of amino acid sequences of Rad, Gem, and N-Ras. Predicted amino acid sequences of Rad, Gem, and Ras are aligned and numbered with the second in-frame methionine of Rad as position one and the first in frame methionine of Rad as position -39. The five G domains are shaded and the variations in positions 61 and 108 are marked. Dashes indicate gaps inserted for optimal alignment of the sequences.
All Ras-related proteins are GTPases.
These GTPases cycle between a GTP-bound (presumably active) form and a
GDP-bound (presumably inactive) form. This cycle is regulated by
proteins that affect GDP dissociation, GTP association, or the rate of
GTP hydrolysis(16) . The former are represented by guanine
nucleotide dissociation inhibitors and guanine nucleotide exchange
factors, the best characterized of which is the mammalian homologue of
the Drosophila Son-of-Sevenless protein (mSos). The latter is
exemplified by GTPase-activating proteins (GAPs). Both
guanine nucleotide exchange factor and GAP activity may be controlled
in response to extracellular stimuli(17) . The role of Rad in
the signaling events is still unknown. However, both Rad and Gem have
marked sequence differences from any known Ras-related proteins in the
effector (G2) domain thought to bind GAP. In the present study we have
expressed Rad protein, characterized its GTP binding and GTPase
activity properties, and studied structure-function relationship by
site-directed mutagenesis and the possible regulation by noncovalent
and covalent modifications.
To assess
the specificity of GTP binding to Rad, the fusion protein (5
µg/lane) was resolved on 10% SDS-PAGE followed by electrotransfer
to nitrocellulose paper (Schleicher & Schuell). The blots were cut
into strips, washed twice with overlay buffer (50 mM Tris-HCl,
pH 7.5, 5 mM MgCl, 1 mM EGTA, 1 mM KH
PO
/K
HPO
, and
0.3% Tween 20), and incubated in the overlay buffer containing 1
µCi/strip of [
-
P]GTP in the absence or
presence of various nucleotides at a concentration of 0.1 mM.
Incubation was continued at room temperature for 60 min, after which
the blots were washed twice for 10-min intervals with the same buffer
supplemented with 20 mM MgCl
. The blots were
air-dried and exposed to X-Omat AR film (Kodak). The amount of GTP was
quantitated by scanning densitometry (Molecular Dynamics).
GST-Rad was also phosphorylated in vitro with the
catalytic subunit of protein kinase A (PKA, Sigma; 20 µg of fusion
protein/25 units of PKA/assay) in a kinase buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl, and 1 mM DTT), and samples were
resolved by SDS-PAGE and autoradiography as described above. When Rad
was to be separated from its fusion partner GST, the phosphorylated
GST-Rad beads were incubated in a cleavage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5 mM CaCl
) containing 0.2 µg thrombin (Sigma) at room
temperature for 60 min(19) .
Figure 2:
Rad is a GTP-binding protein. A,
GST-Rad (1 µg) was incubated with
[-
P]GTP (3 µCi/1.7 µM) in
the absence or presence of various concentrations of Mg
at 22 °C for the indicated time. The calculated free
Mg
concentrations (mM) were:
, 0;
, 0.0005;
, 0.1;
, 1;
, 20 and were produced
by appropriate mixture of MgCl
and EDTA(21) . B, GST-Rad (1 µg) was incubated with
[
-
P]GTP (3 µCi/1.7 µM) or
[
H]GDP (3 µCi/2.2 µM) in the
absence or presence of various concentrations of MgCl
at 22
°C for 10 min or 37 °C for 30 min. The symbols represent GTP
binding (
) and GDP binding (
). C, GST-Rad (5
µg) was resolved by 10% SDS-PAGE, transferred to nitrocellulose
filters, and probed with [
-
P]GTP (1
µCi/lane) in the absence or presence of 0.1 mM of
indicated nucleotides as described under ``Experimental
Procedures.'' D, competition studies were performed as
described for C with individual nitrocellulose strips
incubated with various concentrations of GTP (
) or ATP (
).
Data in A and B are the mean from two or three
separate experiments performed in triplicate (A) or duplicate (B). The data in C and D are the
representative of three separate blots and average of two separate
quantitative determinations.
Figure 3:
Rad is a GTPase. GST-Rad (1 µg) bound
to GSH-Sepharose beads was loaded with
[-
P]GTP (3 µCi) at 22 °C for 10
min. The beads were washed in cold washing buffer, and GTP hydrolysis
preceded at 22 °C for the indicated times (A-C) or
10 min (D) in the absence or presence of various GAPs. The
beads were washed, bound nucleotides were eluted and resolved by
polyethyleneimine cellulose thin layer chromatography as described
under ``Experimental Procedures.'' A shows a typical
autoradiogram representative of three separate experiments. For B-D, the autoradiograms were quantified by scanning
densitometer and the data expressed as percent of GDP in the total
bound guanine nucleotides for two or three separate
experiments.
To better understand the structure-function
relationship of Rad, we performed in vitro site-directed
mutagenesis. Changes in activity of the various mutants with regard to
GTP binding and GTP hydrolysis are summarized in Table 1. As with
Ras itself, a point mutation of Ser to Asn
in
Rad completely abolished its ability to bind GTP (Fig. 4B), indicating the critical role of this residue
in GTP binding or overall structure of the protein. Point mutation of
Pro
to Gly (the amino acid found in Ras) slightly
decreased GTP binding, whereas modification of this residue to Val (a
transforming mutation in Ras) did not result in significant changes in
either GTP binding or hydrolysis. Mutation of Gln
to His
slightly increased GTP binding, but was without effect on GTP
hydrolysis. However, a double mutant, P61V/Q109H, showed extremely high
intrinsic GTPase activity, which was further stimulated to near
complete GTP hydrolysis within 3 min upon addition of as low as 10
µg of total rat liver cytosolic proteins.
Figure 4:
Mutational analysis of Rad. Point
mutations were made using wild type pGEX-2T-rad as template. Each
mutant was confirmed by DNA sequencing. A,
[-
P]GTP binding to wild type (
) and
S66N (
) Rad. Binding was done as described in the legend to Fig. 2, except that 0.25 µg/assay of Rad protein was used. B, [
-
P]GTP hydrolysis by wild type (WT) and P61V/Q109H Rad. GTP hydrolysis was assessed as
described in the legend to Fig. 2. Data are the means of three (A, triplicate) or two (B, single data points)
separate experiments.
Figure 5:
Rad as a phosphoprotein. A,
GST-Rad (20 µg) immobilized on GSH-Sepharose beads was incubated
with the catalytic subunit of PKA (25 units) and
[-
P]ATP (10 µCi) at 30 °C for 15
min. The beads were washed, and GST was cleaved from Rad by thrombin
(0.2 µg) at 22 °C for 60 min. The products, including
phospho-Rad, were then resolved by 10% SDS-PAGE. B, GST-Rad
was phosphorylated as described above and digested with V8 protease at
the indicated concentrations at 37 °C for 1 h after heat
denaturation followed by SDS-PAGE on 15% gel to resolve the
phosphopeptides. C and D, GST-Rad (20 µg)
immobilized on GSH-Sepharose beads was incubated with human skeletal
muscle or L6 cell cytosolic extracts precleared with GST-Sepharose
beads at 4 °C for 90 min. The beads were then subjected to
extensive washings as described under ``Experimental
Procedures'' and incubated with
[
-
P]ATP (10 µCi) at 30 °C for 15
min. Phospho-Rad was detected either by direct 10% SDS-PAGE (C) or cleaved with thrombin followed by electrophoresis (D). The data are the representative of two to five separate
experiments.
We have expressed Rad, a novel Ras-related protein overexpressed in skeletal muscle of humans with type II diabetes, characterized its nucleotide binding and enzymatic activity and regulation by covalent and noncovalent interactions. We find that Rad is a GTP-binding protein and GTPase-regulatable by a specific GAP-like activity and may also be subjected to serine/threonine phosphorylation.
Binding of GTP to Rad is fast and saturable, a characteristic shared
by all Ras-like proteins. For example, like human Ha-Ras p21 (29) and other small G proteins(30) , Rad bound
specifically [-
P]GTP after transfer to
nitrocellulose filters, indicating that in a denatured state Rad still
retains the ability for ligand binding. However, Rad displays several
features that are clearly different from the other members of Ras
family. Thus, N-Ras has a low and linear rate of guanine nucleotide
binding at high (5 mM) magnesium concentrations and increases
its exchange rate at low (0.5 µM) magnesium
concentrations(22) . By contrast, Rad binds minimal amounts of
GTP or GDP at 0.5 µM Mg
, but increases
binding significantly at higher magnesium concentrations. Considering
that intracellular [Mg
] is about 30
mM(31) , with [GTP] far exceeding
[GDP](32) , it is conceivable that Rad may be in a
state of high activity in the normal magnesium concentration in cells.
The unique pattern of Mg dependence may result
from the sequence variation of Rad in the G3 domain (which is known to
be important for interaction with
Mg
/GTP(33) ), since the highly conserved
Ala
and Gly
residues in most other Ras
proteins are replaced with Trp
and Glu
residues in Rad. However, when these two nonconserved amino acid
residues are changed to be identical to those in Ras, the
``Ras-like'' binding characteristics (with regard to
Mg
dependence) are not restored. On the other hand,
like the mutant Ras
GAPs interact with GTPases and greatly accelerate the rate of GTP hydrolysis. Most GAPs are relatively specific in that they exert their effects only on certain subfamilies or subtypes of GTPases. Rad has low intrinsic GTPase activity, and thus, it is reasonable to suggest that to efficiently ``turn off'' Rad in vivo, a GAP must exist. However, Rad is unresponsive to p120 GAP and NF1 which act on Ras(34) , p190 which acts on Rac and Rho GTPases(24) , Rap-GAP(35) , and the recently cloned IQGAP1(26) . Specific Rad-GAP activity, however, is present in all mammalian tissue and cell lines studied. The fact that rat skeletal muscle extract does not show a linear dependence of GAP activity suggests co-expression of other factors that can also affect the GTPase activity of Rad. The ubiquitous distribution of Rad-GAP activity may suggest a role for Rad or Rad-related proteins such as Gem in a variety of cells. At present, the exact nature of Rad-GAP is not known. Preliminary studies have shown that it is a heat-sensitive protein, highly hydrophilic and positively charged at pH 6.0, and elutes in a molecular mass range between 60 and 100 kilodaltons on gel filtration chromatography (data not shown). Purification and characterization studies are under way to identify this GAP.
Although phosphorylation of Ras-like proteins
does not regulate their GTP binding and GTPase activities,
phosphorylation may affect cellular localization. For example, Rap1B in
platelets is translocated from membranes to the cytoplasm when it is
phosphorylated by PKA in response to prostaglandin E and
dibutyryl cAMP stimulation. This also increases the sensitivity of
Rap1B to its guanine nucleotide exchange factor(36) . Rab4 is
serine-phosphorylated by cdc2 kinase in mitotic cells, and this enables
Rab4 to be released into cytosol(37) . Subsequent
dephosphorylation allows reassociation of soluble Rab4 with membranes
upon exit of cells from mitosis. Rad may also be regulated by
phosphorylation. Indeed, Rad is phosphorylated in vitro by PKA
near its carboxyl terminus. The function of this phosphorylation is
unknown, but phosphorylation did not affect Rad's GTP binding or
basal and stimulated GTPase activity (data not shown), consistent with
the effects of phosphorylation on Rap1A, Rap1B, and Rab4. Rad is also
phosphorylated by a kinase that appears to be constitutively associated
with Rad which we have tentatively termed Rad-associated kinase.
Rad-associated kinase is a serine/threonine protein kinase, but is
clearly different from PKA, since it is not stimulated by forskolin or
inhibited by PKA inhibitor. The identification of this kinase activity
may help to elucidate the signal transduction pathway in which Rad is
involved.