In the current study, we found that (1) Tensin2 is present in both murine megakaryocytes and the megakaryocytic leukemia cell line UT7/TPO at the level of specific mRNA, (2) Tensin2 directly interacts with c-Mpl and that Tensin2 is phosphorylated in a TPO-dependent manner and (3) loss of Tensin2 function significantly decreases both TPO-dependent cellular proliferation and Akt signaling. These new findings strongly suggest that the interaction of Tensin2 with c-Mpl is an important event in TPO mediated cellular growth, at least in part through its effect on PI3K/Akt pathway.
Due to the lack of a reliable anti-Tensin2 antibody, we were unable to detect the presence of Tensin2 at the level of the protein. Despite that, the strong evidence of TPO-induced biochemical modifications of Tensin2 as well as Tensin2's biological modulation of cellular proliferation and Akt signaling gives us confidence of the presence and importance of Tensin2 in the megakaryocyte lineage. In addition, we have presented evidence for a potential new path of activation for Akt, i.e., through Tensin2's recruitment of PI3K. Here, we have illustrated the proposed pathway graphically ().
Figure 7 Proposed role of Tensin2 in TPO/c-Mpl signaling of PI3K/Akt. Upon binding of TPO to its receptor, c-Mpl, the intracellular domain of c-Mpl become phosphorylated, one site being at Y631. Tensin2 is then recruited to that site and is phosphorylated. Once (more ...)
In our studies, we showed using two different methods that Tensin2 and c-Mpl directly interact with one another. First, in using the novel peptide microarrays, we were able to identify Tensin2 as a candidate-signaling mediator of TPO effects. Although the use of protein domain microarrays to uncover novel interactions has been previously described in reference 14
, (and MacBeath and Meyer labs unpublished data), our studies are the first in using these assays to expand upon an established pathway, specifically to fill in the details of protein-protein interaction. Secondly, in using a peptide co-precipitation method as the confirmatory method, we have demonstrated not only the two proteins' interaction but also reaffirmed the use of the novel microarrays as an unbiased screening tool to discover new candidates of interaction. Despite numerous attempts, we were unable to consistently show this interaction using co-immunoprecipitation approach. To date, very few direct interactions between SH2 domain proteins and c-Mpl have been reported, a situation most likely due to the known rapid off-rate characteristic of SH2 domain containing proteins and their phosphotyrosine peptide docking sites;5
such binding kinetics make any experiments that require prolonged incubation, such as co-immunoprecipitation assays, problematic for “capturing” such interactions. In fact, the washing steps of the peptide microarrays experiments were rapid for that very reason. Furthermore, co-immunoprecipitation often requires exceptionally high levels of expression of both proteins, which does not pertain for the proteins under study.
Given the reduction of TPO-induced cellular growth and Akt signaling in the Tensin2 knock down experiments, we hypothesize that Tensin2's effect on proliferation is at least partially through its modulation of the Akt pathway, likely by creating a physical link between c-Mpl and the PI3K regulatory subunit, p85. The molecular details by which Tensin2 mediates its functional effects have never been fully described. As we have shown, the likely mechanism in which Tensin2 recruits the PI3K regulatory unit, p85, is by phosphorylation at Y328 of Tensin2's consensus PI3K binding sequence motif YXXM. Although our study focused on TPO-induced modification of Tensin2, it is possible that Tensin2 may be modified in a similar fashion by other receptors and may be a mediator of PI3K/Akt in their pathways.
There have been no studies showing a direct interaction between c-Mpl and PI3K. We (Miyakawa and colleagues) 15 have previously suggested adapter proteins as a mean of PI3K activation when stimulated by TPO. Our current study brings to light this theory and establish Tensin2 as one such potential adaptor. Given that the siRNA-mediated knock down of Tensin2 is unlikely to completely eliminate the protein, our studies argue strongly that Tensin2 may play a significant, but unlikely a solitary role as a mediator between TPO-induced c-Mpl signaling and the PI3K/Akt pathway.
The Tensin family of proteins has previously been described to be involved in focal adhesions and cellular motility, and Tensin2 specifically has been implicated as involved in cellular migration.6,16
In contrast to the results reported here, Tensin2 has also been previously reported to act as a negative regulator of the Akt pathway.10
However, we believe that study was performed in less than ideal conditions to assess for the specific effects on cellular signaling. Unlike the results reported here where we have employed a specific hematopoietic growth factor to assess downstream signaling effects, the study of Hafizi and collegues10
utilized serum, which may have led to multiple effects simultaneously, both inhibitory and stimulatory. In addition, Tensin2 was overexpressed in those cells, which may have disrupted the normal physiologic interactions. Given the use of a loss-of-function approach in our studies as well as the use of a specific ligand with a known receptor, we believe that our studies reflect more focused and physiologic conditions.
It is clear that cellular signal transduction resembles that of a complex electronic circuit with multiple branching and converging pathways. Given the likely large number of mediators in such a complex circuit, candidate gene approach is unlikely to yield all of the components that mediate the specific outcomes of each ligand. Our use of the protein domain microarrays suggests that this approach is an excellent tool in identifying new candidates for further study, especially of those with rapid and transient interactions that have been so difficult to verify previously. For the first time, we have used this novel and unbiased assay to identify a new protein that interacts with c-Mpl, Tensin2. Upon further study of the protein, we have determined that Tensin2's interaction with c-Mpl has a significant effect on TPO-dependent cellular proliferation as well as signaling of the PI3K/Akt pathway. Our work also suggests that Tensin2 may have these effects by directly interacting with PI3K p85 subunit. This work adds to our growing understanding of the molecular signals that mediate thrombopoiesis, but also suggests a novel mechanism of PI3K/Akt activation, which may be relevant in many cellular contexts outside of hematopoiesis and thrombopoiesis.