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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 7, 2005 as doi:10.1096/fj.04-2532fje. |
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Department of Clinical Chemistry, Lund University, Wallenberg Laboratory, University Hospital Malmö, Malmö, Sweden
1 Correspondence: Department of Clinical Chemistry, Lund University, Wallenberg Laboratory, University Hospital Malmö, SE-205 02 Malmö, Sweden. E-mail: sassan.hafizi{at}med.mas.lu.se
SPECIFIC AIMS
We have investigated the effects of overexpression of a novel intracellular molecule, C1 domain-containing phosphatase and TENsin homologue (C1-TEN), on the phenotypic and intracellular signal transduction properties of mammalian HEK293 cells.
PRINCIPAL FINDINGS
1. Stable overexpression of C1-TEN caused a profound alteration in cellular phenotype
Mock transfected cells (M-293) showed an identical morphology to untransfected cells, whereas C1-TEN-transfected cells (C-293) possessed rounder and smoother cell bodies and slightly longer processes. C-293 cells displayed weaker fluorescent staining of filamentous actin. Therefore, C1-TEN overexpression clearly alters cytoskeletal architecture in HEK293 cells.
2. Reduced proliferation and migration of C1-TEN-overexpressing cells
Stable expression of C1-TEN markedly suppressed the growth rate of 293 cells as compared with mock transfection in response to serum over 5 days. Cells overexpressing the active site cysteine mutant C231S showed a fully restored rate of proliferation.
C-293 cells exhibited a markedly reduced rate of migration as compared with M-293 cells (Fig. 1
). After 18 h incubation, C-293 cells had migrated through 8 µm pores toward fibronectin at 50% of the level of M-293 cells.
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3. Serum starvation-induced apoptosis is markedly enhanced by C1-TEN
We wondered whether the inhibited functional properties endowed on C1-TEN-overexpressing cells were linked to a greater rate of apoptosis. Therefore, apoptosis was induced in cells by 5 days incubation in serum-free medium, after which intracellular caspase 3 activity was measured in cell lysates. Serum starvation of C-293 cells resulted in a 3-fold increase in caspase 3 activity as compared with M-293 cells. The continuous presence of the specific peptide caspase 3 inhibitor Z-VAD-FMK blocked the increase in caspase 3 activity, demonstrating specificity of the assay.
4. Inhibition of the Akt intracellular signaling pathway by C1-TEN
We investigated the effect of C1-TEN overexpression on the Akt/PKB signal transduction pathway. Total cell lysates of stably transfected 293 cells were analyzed for their content of specific phosphorylated signaling molecules in the Akt signaling pathway. Relative to total Akt content, all individual clones of C-293 cells contained lower levels of Akt, phosphorylated on Ser473, one of the two phosphorylation sites on Akt that results in its activation. The level of phosphorylation of the Akt substrate GSK3 was also lower in C-293 cells compared with their mock-transfected counterparts. No differences were observed between the cells in their levels of phosphorylation of the transcription factor component c-Jun, a target of the JNK signaling pathway. Therefore, the Akt signaling pathway appears to be specifically suppressed in C1-TEN-expressing cells.
Cell lysates were examined for their levels of Akt kinase enzymatic activity. Endogenous Akt immunoprecipitated from C-293 cell lysates showed a clearly reduced ability to phosphorylate its substrate GSK3 in vitro than Akt isolated from M-293 cells. Therefore, C1-TEN overexpression suppresses Akt enzymatic activity.
5. C1-TEN expression specifically inhibits the Akt, but not ERK, intracellular signaling pathway
To investigate the activation of various signaling pathways in mock vs. C1-TEN-expressing cells, we stimulated these cells with 10% serum and monitored the time courses of Akt and ERK phosphorylation from the same cell lysates. In M-293 cells, serum stimulation caused an increase in Akt phosphorylation after 1 min, which was still elevated at 60 min. In C-293 cells, Akt phosphorylation was delayed in response to serum. A significant increase in Akt phosphorylation was not observed until 5 min after stimulation, which persisted at around the same levels, in contrast to the maximal increase in Akt phosphorylation observed at 30 min in M-293 cells.
The profile of ERK phosphorylation over time in response to serum was distinct from that of Akt. ERK phosphorylation was very rapid, with an increase in pERK levels observed after 1 min and a peak being reached only after 5 min. Phosphorylation of ERK was more transient than that of Akt, as observed by the phosphorylation level dropping after 5 min, although it was still higher than baseline after 60 min. No differences were observed between M-293 and C-293 cells in terms of their time courses of serum-induced ERK phosphorylation. Therefore, C1-TEN expression specifically inhibits the Akt/PKB signaling pathway and not the ERK pathway.
6. Mutation of putative active site Cys231 in C1-TEN restores Akt signaling
The levels of Akt kinase activation and activity in cells overexpressing a potential phosphatase-dead C1-TEN protein were assessed. This construct was produced by mutation of a cysteine at position 231 to a serine, this residue being the equivalent of the catalytic active site cysteine in PTEN (Fig. 2
A). Cell clones expressing mutant C1-TEN (C(C231S)-293) displayed Akt phosphorylation levels comparable to those in mock cells (Fig. 2B
). GSK3 phosphorylation was restored in C(C231S)-293 cells as compared with C-293 cells. Therefore, it is likely that the inhibitory effects of C1-TEN on Akt/PKB signaling are mediated by phosphatase activity.
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CONCLUSIONS AND SIGNIFICANCE
We first identified C1-TEN as a novel intracellular tensin-like protein that was a binding partner to Axl RTK. In the present study, to gain insight into its function, we investigated the biochemical and cellular effects of ectopic C1-TEN expression in HEK293 cells. We observed that C1-TEN affects cells in the same manner as PTEN overexpression (i.e., it enhances proapoptotic pathways and inhibits cell proliferation and migration). C1-TEN specifically inhibited the Akt/PKB signaling pathway, without influencing the ERK signaling pathway.
In addition to the tandem SH2-PTB domains at the C termini of tensin and C1-TEN, both proteins possess a PTPase motif near the N-terminal region. This region in C1-TEN is very similar to the tumor suppressor lipid phosphatase PTEN in terms of 3-dimensional structure, based on the crystal structure of PTEN. The amino acid sequence of C1-TEN and its predicted structural similarities to tensin and PTEN indicate that it may be present at focal adhesions and may possess putative phosphatase activity. The PTB domain of C1-TEN has also been shown to bind to integrin ß subunits. Determining the individual roles of the different domains in the cellular effects of C1-TEN is a focus of current investigation.
Despite the significant similarity of C1-TEN to tensin, several noticeable differences exist, one in the amino acid sequences within their putative PTPase catalytic motifs. The sequence (H/V)C(X5)R(S/T) is a signature motif for PTPases and dual specificity phosphatases, and forms an active site pocket of varying widths and depth. Although neither protein contains all of the residues in this motif, C1-TEN does possess an active site cysteine. This residue is crucial for catalytic activity as it exists as a thiolate anion at neutral pH and forms a thiol-phosphate intermediate in the phosphate hydrolysis reaction that underlies dephosphorylation. The presence of such a cysteine in C1-TEN suggests putative phosphatase activity, which would concur with functions observed in the present study. We investigated this through mutation of Cys231 for Ser and observed restoration of cell proliferation and Akt activation to levels at least as high as in mock cells. This indicates that C1-TEN may be a phosphatase, although the nature of the enzyme and its substrate is unknown. This activity may be of the classical PTPase type and/or of the type displayed by PTEN, which is principally an inositol lipid phosphatase, an activity critical for its role as a negative regulator of the PI3K cascade and consequently its role as a tumor suppressor. In recent years, other "nonclassical" molecules containing variations on the conserved PTPase motif (such as myotubularin) have been characterized and shown to strongly catalyze phosphoinositide hydrolysis.
The domain organization of the C1-TEN molecule indicates that it has the capacity to associate with membrane through either or both the C1 domain and the C2 domain in the PTEN-like region. C1-TEN may bind phosphorylated (SH2 domain) or nonphosphorylated (PTB domain) tyrosine residues in interacting proteins. These protein and membrane interaction propensities may enable C1-TEN to come into close proximity with potential activators and targets in a signaling complex assembled at the membrane (Fig. 3
). For example, the interaction of C1-TEN with Axl RTK may serve to negatively regulate the latter’s links with the PI3K/Akt signal transduction pathway.
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C1-TEN appears to be a novel negative regulator of the Akt/PKB signaling pathway and inhibits cell survival, proliferation, and migration. These effects may be mediated by a putative phosphatase activity in C1-TEN. These properties of C1-TEN therefore have implications for the regulation of signaling originating at RTKs such as Axl, and potentially other molecules such as integrins, leading toward PI3K/Akt signal transduction. These properties of C1-TEN are of potential significance for regulation of cellular processes involved in cancer, atherosclerosis, and inflammation.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2532fje; doi: 10.1096/fj.04-2532fje
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