(Received for publication, August 30, 1995; and in revised form, October 10, 1995)
From the
We have previously reported the tight association of a serine
kinase activity with the human insulin receptor (Lewis, R. E., Wu, G.
P., MacDonald, R. G., and Czech, M. P.(1990) J. Biol. Chem. 265, 947-954). We tested the possibility that the associated
serine kinase activity was intrinsic to the receptor catalytic domain.
The ratio of phosphoserine to phosphotyrosine on insulin receptors
phosphorylated in vitro was used as an index of the associated
serine kinase activity. Phosphorylation and phosphoamino acid analysis
of insulin proreceptors revealed associated serine kinase activity
early in receptor synthesis. Insulin receptors were expressed in HeLa
cells using a recombinant vaccinia virus. The ratio of phosphoserine to
phosphotyrosine on insulin receptors expressed by the recombinant
vaccinia virus was determined relative to endogenous insulin receptors
in cells treated with -amanitin to block host cell mRNA synthesis.
-Amanitin treatment had no effect on the ratio of phosphoserine to
phosphotyrosine on insulin receptors expressed from the recombinant
virus even though they were present in a 4000-fold excess above
endogenous receptors. We conclude that the serine kinase activity
associated with the insulin receptor is intrinsic to the receptor
catalytic domain. Receptor-catalyzed autophosphorylation of serine may
play an important role in modulating insulin receptor signaling.
Insulin-stimulated phosphorylation of the insulin receptor
cytoplasmic domain plays a central role in the transmission and
regulation of insulin receptor signal transduction. Insulin activation
of the receptor tyrosine kinase results in a cascade of tyrosine
autophosphorylation. Autophosphorylation of tyrosines 1158, 1162, and
1163 ()maintain receptor kinase activation in the absence of
bound insulin(1, 2) . Phosphorylation of tyrosines
1328 and 1334 in the receptor carboxyl-terminal tail occurs upon
activation of the insulin receptor tyrosine
kinase(3, 4, 5) . Serine phosphorylation of
the insulin receptor cytoplasmic domain also occurs following insulin
activation but subsequent to receptor tyrosine
autophosphorylation(6, 7) . Phorbol ester or forskolin
addition to cells has been reported to stimulate the serine
phosphorylation of the insulin receptor
subunit(8, 9, 10) . A decrease in insulin
receptor tyrosine kinase activity coincides with the phosphorylation of
insulin receptors by these agents(10, 11) . Protein
kinase C (12) and cyclic AMP-dependent protein kinase (13) are each capable of phosphorylating the insulin receptor
in cell-free systems. Receptor phosphorylation catalyzed by each of
these kinases results in a decrease in insulin-stimulated tyrosine
kinase activity toward exogenous substrates(12, 13) .
Additional studies with kinase-inactive insulin receptor mutants
indicate that the receptor tyrosine kinase is important for the
complete biological activity of insulin. Consequently, inhibition of
receptor tyrosine kinase activity by serine phosphorylation may be an
important mechanism for regulating receptor signaling in intact cells.
An insulin-sensitive serine kinase activity is associated with the insulin receptor in eluates from wheat germ-agglutinin affinity columns(14, 15, 16) . This insulin-stimulated serine kinase activity remains tightly associated with highly purified insulin receptors eluted from insulin-agarose affinity columns(17, 18) . The tightly associated serine kinase activity is also capable of phosphorylating highly purified insulin receptors on serine and threonine sites within the cytoplasmic domain that are also phosphorylated in intact cells (17) . The serine kinase activity tightly associated with the insulin receptor phosphorylates synthetic peptides identical to sites of insulin receptor serine and threonine phosphorylation in vivo. The conclusion that the insulin receptor activates the receptor-associated serine kinase is supported by the observation that peptide phosphorylation is enhanced by insulin addition to the affinity-purified receptor preparations(17) .
The presence of serine kinase activity associated with insulin receptors immunoprecipitated from Sf9 cells infected with a recombinant baculovirus containing the human insulin receptor cDNA has been reported(6) . Although these receptor preparations contain primarily unprocessed proreceptor that is unresponsive to insulin activation, they suggest the possibility that the insulin receptor may contain intrinsic serine/threonine as well as tyrosine kinase activity. Consistent with this possibility, the tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid blocks the ability of insulin receptor preparations from baculovirus-infected Sf9 cells containing associated serine kinase activity from phosphorylating a synthetic peptide containing an insulin receptor serine phosphorylation site(6) .
Biochemical analysis has provided evidence for the existence of several ``dual specificity'' kinases(19) . The amino acid sequences of those kinases considered to be capable of phosphorylation on tyrosine, serine, and threonine most closely resemble the family of serine/threonine kinases. We tested the hypothesis that the insulin receptor kinase had the capability of phosphorylating its cytoplasmic domain on serine and tyrosine. We expressed the insulin receptor under control of the bacteriophage T7 promoter in HeLa cells that were also infected with a recombinant vaccinia virus that expresses T7 polymerase. Receptor-associated serine kinase activity can be detected in insulin proreceptors early in synthesis. Furthermore, we demonstrate that the serine kinase activity associated with insulin receptors expressed under control of the T7 promoter is not altered in receptor preparations isolated under conditions that block synthesis of host cell mRNA.
The purpose of this study was to test whether insulin
receptor-associated serine kinase activity is intrinsic to the
receptor. To test this possibility, human insulin receptor was
initially expressed in COS-1 cells. Extracts from transfected cells
were partially purified on wheat germ agglutinin-agarose. The
flow-through fraction from the wheat germ agglutinin-agarose was
applied to lentil lectin-agarose. Insulin receptors bound to the lectin
affinity resins were eluted and then immunoprecipitated with the
anti-insulin receptor antibody CT-1(24) . Insulin receptors
were phosphorylated in the immune complex by incubation with
[-
P]ATP for 30 min and then analyzed by
denaturing polyacrylamide gel electrophoresis (Fig. 1A). Insulin receptor antibodies precipitated two
proteins from the wheat germ agglutinin-agarose eluate that could be
phosphorylated in the immune complex. The autoradiogram in Fig. 1A demonstrates phosphoproteins of 200 and 97 kDa
that represent the uncleaved insulin receptor precursor and the mature
insulin receptor
subunit, respectively.
Figure 1:
Phosphorylation of insulin receptors
isolated from transfected COS-1 cells by lectin purification. A, insulin receptors were partially purified from transfected
COS-1 cells on wheat germ agglutinin-agarose (WGA, left
lane) or by collecting the flow through from the WGA column and
applying it to lentil lectin-agarose (LL, right
lane). Bound insulin receptors were eluted from each column and
precipitated with antibody 83-14 that recognizes the insulin
receptor subunit. Receptors were phosphorylated in the immune
complex with [
-
P]ATP, washed, and separated
by electrophoresis on an 7% polyacrylamide gel.
P-labeled
receptors were visualized by autoradiography of the gel. B,
phosphoamino acid analysis of insulin receptors phosphorylated
following isolation on lectin columns. Insulin proreceptors and mature
insulin receptor
subunits isolated in A were hydrolyzed
and
P-labeled phosphoamino acids were separated
electrophoretically in two dimensions on thin layer cellulose plates.
The relative migration of ninhydrin-stained phosphoamino acid standards
is indicated (S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine) in the upper left hand
panel.
Biosynthetic labeling
studies demonstrated that the insulin receptor is initially synthesized
in the endoplasmic reticulum as a high mannose proreceptor
(190-210 kDa) that undergoes subsequent maturation of its N-linked carbohydrate side chains and proteolytic processing
to produce mature and
subunits(26, 27, 28) . To isolate insulin
receptor precursors in the endoplasmic reticulum containing high
mannose carbohydrates, we incubated the flow-through from wheat germ
agglutinin-agarose with lentil lectin-agarose. The lentil
lectin-agarose eluate was immunoprecipitated with antibody CT-1 as
described above. Phosphorylation of the immune complex and
SDS-polyacrylamide gel electrophoresis revealed a single phosphoprotein
of approximately 200 kDa (Fig. 1A). This phosphoprotein
most likely represents high mannose insulin receptor
monomer
isolated from early in the synthetic pathway.
Phosphorylation of
insulin proreceptors and mature insulin receptor subunits
immunoprecipitated from wheat germ agglutinin-agarose revealed not only
phosphotyrosine but also phosphoserine (Fig. 1B, left panels). Interestingly, serine phosphorylation was also
detected in high mannose insulin receptor precursors (Fig. 1B, right panel). Trace amounts of
phosphothreonine could also be detected in receptor precursors
immunoprecipitated from wheat germ agglutinin-agarose or lentil
lectin-agarose eluates (Fig. 1B). The phosphoserine
content of receptor precursor and
subunit immunoprecipitated from
wheat germ agglutinin-agarose was 11-12% of the phosphotyrosine
released by hydrolysis from these phosphoproteins (Table 1).
Similarly, phosphoserine detected in proreceptor from lentil
lectin-agarose eluates was approximately 15% of detectable
phosphotyrosine (Table 1).
The data in Fig. 1suggest
that serine kinase activity becomes associated with the insulin
receptor early in its biosynthetic pathway. The level of serine
phosphorylation detected with the high mannose form of the insulin
receptor precursor appears comparable to that co-precipitating with
biosynthetically mature forms. These observations suggest that either a
distinct serine kinase associates with insulin receptor precursors
early in synthesis or the insulin receptor tyrosine kinase has the
intrinsic ability to phosphorylate itself on serine residues. To
distinguish between these two possibilities, we developed a system to
express the insulin receptor in cells in which endogenous mRNA
synthesis had been blocked for prolonged periods of time. We subcloned
the insulin receptor cDNA into the transfer vector pTM-1 (21) to create the plasmid pTM1hIR. This construct contains a
T7 promoter and 5`-untranslated sequences from encephalomyocarditis
virus for efficient transcription and translation of uncapped insulin
receptor mRNA. Insulin receptors were detected by immune complex kinase
assay of insulin receptor immunoprecipitates from HeLa cells
transfected with pTM1hIR and infected with a recombinant vaccinia virus
(VTF7) encoding T7 polymerase (Fig. 2A, lane
5). To determine the amount of endogenous insulin receptor
contributed by HeLa cells, control infections and transfections were
performed. Similar amounts of phosphorylated insulin receptor precursor
and insulin receptor subunit were detected in the immune complex
from HeLa cells infected with wild-type vaccinia virus (WR) and
transfected with recombinant plasmid pTM1HIR (Fig. 2A, lane 1) or when infected with the recombinant virus VTF7 and
transfected with the control plasmid pTM-1 (Fig. 2A, lane 2).
Figure 2:
The effect of -amanitin on T7
polymerase-mediated expression of insulin receptors in HeLa cells. A, insulin receptors were immunoprecipitated from HeLa cells
infected with a control vaccinia virus (WR; lanes 1 and 3) or a recombinant vaccinia virus (VTF7; lanes 2, 4, 5, and 6) that expresses bacteriophage T7
polymerase. Virus-infected cells were transfected with a control
plasmid (pTM-1; lanes 2 and 4) or the same plasmid
containing the human insulin receptor cDNA (pTM1hIR; lanes 1, 3, 5, and 6). Virus-infected and transfected
cells were incubated without (lanes 1, 2, and 5) or with (lanes 3, 4, and 6) 10
µg/ml
-amanitin for 18 h at 37 °C and lysed, and the
insulin receptors were immunoprecipitated with antibody 83-14
following partial purification on wheat germ agglutinin-agarose.
Receptors were phosphorylated in the immune complex with
[
-
P]ATP, washed, and separated by
electrophoresis on an 7% polyacrylamide gel.
P-labeled
receptors were visualized by autoradiography of the gel.
Autoradiography of lanes 1-4 was for 2 h.
Autoradiography of lanes 5 and 6 was for 30 min. B, phosphoamino acid analysis of insulin receptors isolated
from pTM1hIR-transfected cells. Insulin proreceptors and mature insulin
receptor
subunits isolated in A were hydrolyzed and
P-labeled phosphoamino acids were separated
electrophoretically in two dimensions on thin layer cellulose plates.
The relative migration of ninhydrin-stained phosphoamino acid standards
is indicated (S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine) in the upper left hand
panel.
Insulin receptors translated from T7-generated
transcripts could be distinguished from insulin receptors derived from
endogenous HeLa mRNA by the addition of the RNA polymerase II inhibitor
-amanitin(29) . Treatment of control cells with 10
µg/ml
-amanitin for 18 h prior to lysis blocked endogenous
insulin receptor synthesis (Fig. 2A, lanes 3 and 4).
-Amanitin had no effect, however, on the
level of insulin receptor expressed in cells infected with VTF7 and
transfected with pTM1hIR (Fig. 2A, lane 6). In
multiple experiments insulin receptors expressed in VTF7-infected and
pTM1hIR-transfected cells were 8-140-fold more abundant than
endogenous insulin receptors from control cells.
Phosphoamino acid
analysis was performed on insulin receptors from control-treated HeLa
cells and from HeLa cells infected with VTF7 and transfected with
pTM1hIR. Quantitative analysis of the P incorporated on to
tyrosine and serine in the insulin receptor
subunit demonstrated
that treatment with
-amanitin has no effect on the relative amount
of serine phosphorylation of insulin receptors expressed from T7
polymerase-generated transcripts (Fig. 2B and Table 1). Thus, serine kinase activity remained associated with
the insulin receptors synthesized from T7 polymerase-generated
transcripts even though endogenous mRNA synthesis was inhibited.
The
efficiency of insulin receptor production was improved by the
construction and co-infection of a recombinant vaccinia virus, VTF7hIR,
that contained the human insulin receptor cDNA under control of the T7
promoter. Co-infection of HeLa cells with VTF7 and VTF7hIR followed by
treatment with -amanitin resulted in a 4000-fold increase in
mature, phosphorylated insulin receptors over the level of receptor
observed in
-amanitin-treated cells infected with VTF7 and the
control virus WR (Fig. 3, A and B).
Phosphoamino acid analysis of
P-labeled insulin receptors
revealed that phosphoserine persisted in both the proreceptor and
mature
subunit produced in VTF7hIR-infected and
-amanitin-treated cells (Fig. 3C). Quantitative
analysis of phosphoamino acids present in the
P-labeled
insulin receptors produced in VTF7hIR-infected and
-amanitin-treated cells revealed phosphotyrosine:phosphothreonine
ratios of 6.3 and 10.7 in the proreceptors and mature
subunits,
respectively. These ratios are comparable to those seen at lower levels
of insulin receptor expression in COS-1 cells and in
pTM1hIR-transfected HeLa cells ( Fig. 4and Table 1).
Figure 3:
Vaccinia virus-mediated expression of the
insulin receptor in -amanitin-treated cells. A,
P-labeled insulin receptors isolated from insulin-treated
HeLa cells were co-infected with vaccinia viruses WR and VTF7 (lanes 1 and 2) or VTF7 and VTF7hIR (lane
3). Following infection, cells were treated with
-amanitin (lanes 2 and 3) or left untreated (lane 1).
P-labeled proteins were resolved by SDS-polyacrylamide gel
electrophoresis and visualized on a Molecular Dynamics PhosphorImager.
For purposes of clarity, the intensity of lane 3 was decreased
100-fold to approximate that of lanes 1 and 2. B, the relative expression of
P-labeled insulin
receptor
subunits and proreceptors isolated from virus-infected
cells. Expression was normalized relative to the amount of
phosphorylated
subunits (left panel) or phosphorylated
proreceptors (right panel) detected in A (lane
1). C, phosphoamino acid analysis of
P-labeled insulin receptors produced in HeLa cells
infected with VTF7hIR. Insulin proreceptor and insulin receptor
subunit from lane 3 in A were analyzed for
phosphoamino acid analysis as described under ``Experimental
Procedures.''
Figure 4:
Serine and threonine phosphorylation of
insulin receptors expressed at different levels in transfected or
virus-infected cells. Insulin receptors expressed in HeLa cells were
immunoprecipitated and phosphorylated in vitro with
[-
P]ATP. Insulin proreceptors (lanes
1-7) and insulin receptor
subunits (lanes
8-12) were isolated and phosphorylated from HeLa cells
transfected with plasmids pTM1hIR (lanes 1, 3, 5, 6, 8, 10, and 11) or
control plasmid pTM-1 (lanes 2, 4, and 9)
and infected with the wild-type control vaccinia virus WR (lanes
1, 3, and 8) or the recombinant vaccinia virus
VTF7 (lanes 2, 4, 5, 6, 9, 10, and 11). Insulin receptor expression was also
generated by co-infection with the recombinant viruses VTF7hIR and VTF7 (lanes 7 and 12). Following transfection and
infection procedures, cells were incubated with (lanes 3, 4, 6, 7, 11, and 12) or
without (lanes 1, 2, 5, 8, 9, and 10)
-amanitin for 18 h.
P-labeled insulin receptor levels in proreceptors (lanes 2-6) and in
subunits (lanes
9-11) were normalized to control treatments in lanes 1 and 8, respectively.
P-labeled insulin
receptor levels indicated in lanes 7 and 12 were
normalized relative to receptor expression in cells co-infected with
control viruses WR and VTF7 (Fig. 3). The ratio of
phosphotyrosine to phosphothreonine was determined from phosphoamino
acid analysis of each receptor sample and is indicated as an open
circle. The presence of multiple circles denotes additional
experiments for that condition.
Phosphopeptide mapping was performed to determine if treatments that
inhibited the synthesis of endogenous insulin receptors specifically
altered the phosphorylation of individual sites within the insulin
receptor cytoplasmic domain. Hela cells co-infected with VTF7 and
VTF7hIR were treated with -amanitin or left untreated. Insulin
receptors were immunoprecipitated, labeled with
[
-
P]ATP in the presence of 100 nM insulin, and resolved by SDS-polyacrylamide gel electrophoresis.
P-labeled receptor
subunits were localized by
autoradiography and digested with trypsin. The resulting
phosphopeptides were resolved by HPLC into three sets of peaks (Fig. 5A). Two-dimensional analysis revealed that all
phosphopeptides were conserved between insulin receptors isolated from
-amanitin-treated and untreated cells. Furthermore, three
phosphopeptides within HPLC peak 2 contained phosphoserine and were
conserved between
-amanitin-treated and untreated cells (Fig. 5, B and C). All other phosphopeptides
resolved in each HPLC peak contained only phosphotyrosine (data not
shown). These data demonstrate that although
-amanitin treatment
blocks endogenous insulin receptor expression, it has no effect on the
ability of over-expressed insulin receptors to be phosphorylated on
specific serine phosphorylation sites in vitro.
Figure 5:
Phosphopeptide mapping of insulin
receptors isolated from VTF7hIR-infected cells. A, HeLa cells
infected with VTF7 and VTF7hIR were treated with or without
-amanitin, labeled with
P, and treated with insulin
as described in the legend to Fig. 3. Phosphopeptides from
P-labeled receptor
subunits were separated by HPLC,
and individual fractions were counted for radioactivity. B,
phosphopeptides within HPLC peak 2 from insulin receptor treated with
and without
-amanitin were analyzed in two dimensions on thin
layer plates. The location of the
P-labeled
phosphopeptides was determined by autoradiography of the thin layer
plates. C, phosphoamino acid analysis of phosphopeptides
marked A, B, and C in panel B. The
relative migration of ninhydrin-stained phosphoamino acid standards is
indicated.
We investigated the nature of the insulin receptor-associated
serine kinase activity by examining its temporal association with the
insulin receptor during synthesis. Our observations indicate that
insulin receptor serine kinase activity is detectable in insulin
receptors at early stages of receptor synthesis and persists during
treatments that block endogenous mRNA expression. Serine kinase
activity was detected in high mannose forms of the insulin proreceptor,
a form of the receptor found in the endoplasmic reticulum and medial
Golgi(26, 27) . The extent of proreceptor serine
phosphorylation was comparable to that associated with fully processed
receptor subunits (Fig. 1B and Table 1). If
a distinct serine kinase associates with the insulin receptor, this
observation suggests that it must do so prior to maturation of
carbohydrate side chains and proteolytic processing of receptor
subunits.
We also determined whether serine kinase activity would
remain associated with insulin receptors translated from receptor mRNA
transcripts generated by T7 polymerase in cells treated with and
without 10 µg/ml -amanitin for 18 h.
-Amanitin blocks de novo mRNA synthesis by inhibiting RNA polymerase
II(29) .
-Amanitin has no effect however, on T7
polymerase. Thus,
-amanitin treatment of cells infected with VTF7
and transfected with pTM1hIR will yield insulin receptors produced only
by T7 polymerase provided by the recombinant virus (Fig. 2A, compare lanes 5 and 6).
Insulin receptors expressed in VTF7-infected and pTM1hIR-transfected
cells were at least 8-fold more abundant than insulin receptors in
control cells (note times of exposure for lanes 5 and 6
versus lanes 1-4 in Fig. 2A).
Insulin
receptor levels were elevated greatly with a recombinant vaccinia
virus, VTF7hIR, encoding the human insulin receptor under control of
the T7 promoter. Co-infection of VTF7hIR with the virus VTF7 allowed
T7-mediated expression of insulin receptors in -amanitin-treated
cells that was 4000-fold above endogenous receptor expression in
-amanitin cells infected with control virus (Fig. 3).
Despite the ability of
-amanitin to block endogenous insulin
receptor production, VTF7hIR-generated insulin receptors retained
serine kinase activity comparable to that of endogenous insulin
receptors isolated from control-treated cells (Fig. 3C and 4). The fact that the amount of serine phosphate on the
insulin receptor precursor and mature
subunit does not change
appreciably in cells over-expressing the receptor could be explained by
the presence of an excess amount of an associating serine kinase.
However, persistence of insulin receptor-associated serine kinase
activity of VTF7/pTM1hIR cells treated with
-amanitin for 18 h
would mean that a distinct serine kinase activity would also have an
extremely slow turnover rate compared with the endogenous insulin
receptors that were abolished by
-amanitin. Consequently, we
believe a more likely explanation for the insulin receptor-associated
serine kinase is that the kinase domain of the insulin receptor
subunit has the intrinsic ability to transfer phosphate to serine as
well as tyrosine. Phosphopeptide mapping and phosphoamino acid analysis (Fig. 5) demonstrated that the pattern of serine phosphorylation
on insulin receptors in VTF7hIR-infected cells is not altered by
-amanitin treatment. Thus, the serine kinase activity associated
with the insulin receptor would not appear to be composed of multiple
activities both distinct and intrinsic to the insulin receptor
catalytic domain.
Previous reports have demonstrated that an
insulin-sensitive serine kinase activity was associated with human
insulin receptors partially purified from human placental membranes on
wheat germ
agglutinin-agarose(14, 15, 16, 17) .
An associated serine kinase is reportedly dissociated from the insulin
receptor by 1 M NaCl (15, 30) , and that
replacement of the NaCl eluate could reconstitute serine
phosphorylation of the receptor. Other investigations found that a
serine kinase activity remained associated with insulin receptors
purified with 1 M NaCl included in the chromatography
buffer(17, 18) . We demonstrated previously that
affinity-purified preparations of insulin receptor retained an
insulin-stimulated serine kinase activity that phosphorylated the
insulin receptor at serines 1293 and 1294(17) . Our observation
that affinity-purified receptor preparations could also phosphorylate
synthetic peptides identical to sequences containing serines 1293/1294
indicated that the associated serine kinase activity was activated in
response to insulin. However, this serine kinase activity associated
with the insulin receptor was not capable of phosphorylating peptide
substrates of other known kinases(17) . The associated serine
kinase activity could not be dissociated from the insulin receptor by
gel filtration or density gradient centrifugation. ()These
data suggest that there may be at least two serine kinase activities
associated with the insulin receptor. One of these kinases may be a
distinct enzyme separable upon treatment with high salt concentrations.
The data presented here suggest that at least one component of the
insulin receptor-associated kinase activity is intrinsic to the insulin
receptor. Our data indicate that the insulin receptor is a dual
specificity kinase (19) capable of phosphorylating serine as
well as tyrosine residues. Previous experiments with the tyrosine
kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid support
this conclusion(6) .
The majority of eukaryotic protein kinases can be classified as protein serine/threonine kinases or protein tyrosine kinases. The predicted amino acid sequences of cloned kinase genes demonstrate structural differences that aid in this categorization. Several proteins with overall homology that is closer to the serine/threonine family of protein kinases have been shown to be able to autophosphorylate serine, threonine, and tyrosine residues. These dual specificity kinases may form a distinct family of enzymes, and their primary amino acid sequences have been suggested to contain information predictive of their catalytic specificity(19) . The insulin receptor, however, shares greater homology with protein tyrosine kinases than with dual specificity kinases. This difference demonstrates the importance of directly determining phosphoacceptor specificity.
Phosphorylation of the insulin receptor by the associated serine kinase occurs on sites within the juxtamembrane domain (31, 32) and the carboxyl-terminal tail(17) . Phosphopeptide maps suggest that as many as four additional serine phosphorylation sites for the receptor-associated serine kinase may exist(17) . Determination of the role of serine autophosphorylation in the enzymatic function of the insulin receptor and in insulin-mediated control of cellular metabolism may require identification of these sites. Analysis of mutant receptors lacking all serine phosphorylation sites may be necessary to evoke an effect on receptor enzymatic activity and signaling.