(Received for publication, March 20, 1996)
From the
Insulin stimulation of Chinese hamster ovary cells expressing
the human insulin receptor and differentiated 3T3L1 adipocytes resulted
in a time-dependent reduction in the SDS-polyacrylamide gel
electrophoretic mobility of STAT3. The decreased STAT3 mobility
initially occurred by 2 min and was quantitative by 5 min. In addition,
the change in STAT3 mobility was concentration-dependent and was
detectable at 0.3 nM insulin with maximal effect between 1 and
3 nM. Although both these cell types also express the
STAT1, STAT1
, STAT5, and STAT6 isoforms, only STAT3 was
observed to undergo an insulin-dependent reduction in mobility.
Immunoprecipitation of STAT1 and STAT3 from
P-labeled
cells demonstrated that only STAT3 was phosphorylated in response to
insulin whereas phosphoamino acid analysis indicated that this
phosphorylation event occurred exclusively on serine residues.
Furthermore, treatment of cell extracts with alkaline phosphatase
reversed the insulin-stimulated decrease in STAT3 mobility. Together,
these data demonstrate that insulin is a specific activator of STAT3
serine phosphorylation without affecting the other STAT isoforms.
Signal Transducers and Activators of Transcription (STAT) ()proteins have emerged as important biological links
between cell surface receptors and DNA transcription events (reviewed
in (1, 2, 3, 4) ). Currently, there
are six general members of this growing protein family (STAT1-6)
with additional subtypes for several of these
members(5, 6, 7) . These proteins were first
identified as Src Homology 2 (SH2) and Src Homology 3 (SH3)
domain-containing tyrosine-phosphorylated substrates of cytokine
receptor signaling pathways(8) . It is now generally accepted
that activation of various cytokine receptors results in the
stimulation of the Janus kinase family (JAK1, -2, and -3 and Tyk2) of
protein tyrosine kinase(4) . The activated JAK kinases directly
tyrosine phosphorylate the STAT proteins, which induce both hetero- and
homodimerization through phosphotyrosine recognition by the STAT SH2
domains(9, 10) . The STAT protein dimers then
translocate into the nucleus and bind to specific DNA recognition
sequences resulting in increased transcriptional activation of various
effector
genes(7, 11, 12, 13, 14, 15, 16) .
In addition to cytokine receptors, several growth factor tyrosine
kinase receptors (epidermal growth factor, platelet-derived growth
factor, and colony-stimulating factor) as well as non-tyrosine kinase
receptors (prolactin, growth hormone, and angiotensin II) also induce
the tyrosine phosphorylation, dimerization, and transcriptional
activation of the STAT
proteins(12, 17, 18, 19, 20, 21, 22, 23) .
However, since insulin does not stimulate the JAK kinases or increase
the tyrosine phosphorylation of the STAT proteins, it is generally
accepted that insulin does not impinge upon this particular signal
transduction pathway(18, 24, 25) .
Nevertheless, in contrast to tyrosine phosphorylation, recent studies
have indicated that growth hormone, interferon , and interleukin 6
can also stimulate the serine phosphorylation of the STAT proteins in a
manner distinct from the JAK-mediated tyrosine
phosphorylation(11, 26, 27, 28) .
Furthermore, both tyrosine and serine phosphorylation of STAT1 and
STAT3 is required for maximal DNA transcriptional activity of reporter
genes presumably through fostering STAT
homodimerization(28, 29) . Based upon these findings,
we have re-evaluated the potential role of insulin on STAT
phosphorylation. In this study, we demonstrate that insulin stimulation
results in a rapid quantitative serine phosphorylation of STAT3 without
any significant effect on STAT1
, STAT1
, STAT5, or STAT6
phosphorylation.
Figure 1: Insulin stimulates a decrease in SDS-polyacrylamide gel electrophoretic mobility of the STAT3 protein. CHO/IR and 3T3L1 adipocytes were either unstimulated (C, lanes 1 and 3) or stimulated for 5 min with 100 nM insulin (I, lanes 2 and 4) at 37 °C. Whole cell detergent extracts were prepared, and 50 µg were subjected to SDS-polyacrylamide gel electrophoresis and Western blotted with STAT1 (A), STAT3 (B), STAT5 (C), or STAT6 (D) antibodies as described under ``Experimental Procedures.'' IB, immunoblot.
Nevertheless, insulin stimulation had no effect
on the apparent mobility of STAT1, STAT1
, STAT5, and STAT6 (Fig. 1, A, C, and D). In contrast,
insulin treatment for 5 min of either CHO/IR or 3T3L1 adipocytes
resulted in a marked reduction of SDS-polyacrylamide gel
electrophoretic mobility of STAT3 (Fig. 1B). This
insulin-stimulated decrease in STAT3 SDS-polyacrylamide gel
electrophoretic mobility was a typical characteristic of
post-translational serine/threonine phosphorylation similar to that
observed for a number of intracellular signaling proteins including
STAT3(32, 33, 34) .
Figure 2: Time and concentration dependence of the insulin-stimulated decrease in STAT3 electrophoretic mobility. CHO/IR (A) or 3T3-L1 adipocytes (B) were incubated in the absence or the presence of 100 nM insulin for the times indicated at 37 °C. Whole cell detergent extracts (150 µg) were prepared and subjected to Western blotting using a STAT3 antibody as described under ``Experimental Procedures.'' CHO/IR cells (C) were incubated for 5 min at 37 °C with various concentrations of insulin as indicated. Whole cell detergent extracts were prepared and Western blotted with the STAT3 antibody as described above.
Figure 3:
Insulin stimulates the serine
phosphorylation of STAT3. A, CHO/IR cells were labeled with P and incubated in the absence (C, lanes 1 and 3) or presence of 100 nM insulin (I, lanes 2 and 4) for 5 min at 37 °C. Whole cell detergent
extracts were prepared and immunoprecipitated using the STAT3 antibody (lanes 1 and 2) or the STAT1 antibody (lanes 3 and 4) as described under ``Experimental
Procedures.'' The immunoprecipitates (IP) were then
resolved by SDS-polyacrylamide gel electrophoresis, transferred to PVDF
filter membranes, and subjected to autoradiography. Unlabeled CHO/IR
cells were treated identically as described above and
immunoprecipitated with the STAT3 antibody (lanes 5 and 6) or the STAT1 antibody (lanes 7 and 8) and
subjected to STAT3 and STAT1 Western blotting as indicated. IB, immunoblot. B, the immunoprecipitated
P-labeled STAT3 bands from control and insulin-stimulated
cells described in A were cut from the PVDF filter membranes
and subjected to one-dimensional phosphoamino acid analysis.
Phosphoamino acid standards are indicated. P-Ser,
phosphoserine; P-Thr, phosphothreonine; P-Tyr,
phosphotyrosine. C, 150 µg of cell lysate harvested from
CHO/IR cells either untreated (C) or incubated with 100 nM insulin for 5 min (I) were subjected to treatment with
alkaline phosphatase (PPase, 1000 units/ml) for 1 h at room
temperature. The samples were resolved by SDS-polyacrylamide gel
electrophoresis and Western blotted with a STAT3
antibody.
Previous studies have reported that insulin neither activates the JAK kinases nor stimulates the tyrosine phosphorylation of STAT proteins (18, 24, 25) . Consistent with these data, we were unable to detect any tyrosine phosphorylation of STAT3 by phosphotyrosine immunoblotting (data not shown). Thus, we next performed phosphoamino acid analysis of STAT3 isolated from unstimulated and insulin-stimulated cells (Fig. 3B). These data directly demonstrate that insulin stimulates the serine phosphorylation of STAT3 without any significant tyrosine or threonine phosphorylation.
Even though the insulin-stimulated serine phosphorylation of STAT3 correlated with its reduction of electrophoretic mobility, this does not prove cause and effect. Thus, to confirm that the serine phosphorylation of STAT3 was responsible for the reduction in mobility, extracts from unstimulated and insulin-stimulated cells were treated with alkaline phosphatase (Fig. 3C). As previously observed, STAT3 displayed the characteristic decreased mobility following insulin stimulation (Fig. 3C, lanes 1 and 2). Alkaline phosphatase treatment of extracts from unstimulated cells did not alter the mobility of STAT3 (Fig. 3C, lane 3). In contrast, alkaline phosphatase treatment of extracts from insulin-stimulated cells displayed an increase in mobility to the same extent as STAT3 from control cells (Fig. 3C, lane 4). These results demonstrate that the insulin-stimulated serine phosphorylation of STAT3 was directly responsible for the reduction in SDS-polyacrylamide gel electrophoretic mobility.
In summary,
insulin-stimulated serine phosphorylation of STAT3 is unique in several
ways compared with other hormones that activate the STAT pathway. For
example, ciliary neurotrophic factor, oncostatin M, leukemia-inhibitory
factor, interleukin 6, growth hormone, and interferon have all
been found to induce both tyrosine and serine phosphorylation of STAT
proteins(11, 28, 29, 35) . However,
the data presented in this study demonstrate that insulin is the only
ligand to date that mediates the exclusive serine phosphorylation of a
STAT protein. Although it has recently been suggested that both
tyrosine and serine phosphorylation were due to a bifurcation of the
JAK pathway (27) , our data indicate the presence of at least
two distinct tyrosine and serine kinase pathways. Furthermore, our data
demonstrate that the insulin-stimulated serine kinase pathway
responsible for STAT protein phosphorylation was apparently specific
for STAT3. This supports previous reports, which demonstrated that the
interleukin-6 receptor selectively serine-phosphorylated STAT3 but not
STAT1
(36) . Currently, the identification of the serine
kinase and molecular pathways mediating these differences remains to be
determined.