Pleckstrin homology (PH) domains play vital roles in enabling proteins to recognise phosphoinositides. Simon Dowler (Dario Alessi's 2nd PhD Student) identified two proteins over 10 years ago now termed the Tandem PH containing Protein-1 (TAPP1) and TAPP2. These are related adaptor proteins consisting of two sequential PH domains in which the C-terminal PH domain binds with high affinity to PtdIns(3,4)P2¬ [1]. Despite intensive analysis of many PH domain containing proteins, TAPP1 and TAPP2 to our knowledge are the only proteins known to specifically interact with PtdIns(3,4)P2 with high affinity.
Christine Milburn (Dario Alessi's 3rd PhD Student, jointly supervised by Daan van Aalten) solved the structure of the C-terminal PH domain of TAPP1 revealing the mechanism by which it recognised PtdIns(3,4)P2 specifically [2]. Christine demonstrated that mutation of residues, Arg211 on TAPP1 or the equivalent Arg218 residue in TAPP2, that interacted with PtdIns(3,4)P2, completely prevented interaction of these proteins with PtdIns(3,4)P2 [2]. Interestingly, Christine structural data also suggested that binding of TAPP1 to PtdIns(3,4,5)P3 was inhibited by steric hindrance of an Ala residue located close to the position in which the 5'-phosphate would be expected to reside. The equivalent residue in PtdIns(3,4,5)P3-binding PH domains is frequently a Gly. Mutation of the Ala residue to Gly in TAPP1 resulted in it being capable of interacting with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity [2].
Wendy Kimber (Dario Alessi's 6th PhD Student) next provided the first evidence suggesting that TAPP1 binds PtdIns(3,4)P2 selectively in vivo, by showing that TAPP1 relocated from the cytosol to the plasma membrane of cells, following stimulation with agonists that induced PtdIns(3,4)P2, but not with those that induced mainly PtdIns(3,4,5)P3 [3]. Aaron Marshall's group in Winnipeg also demonstrated in B cells, both TAPP1 and TAPP2 translocated to the plasma membrane in response to antigen stimulation and this correlated with the formation of PtdIns(3,4)P2 rather than production of PtdIns(3,4,5)P3 [4].
Despite this work the biological functions of TAPP1 and TAPP2 remained elusive. Apart from the PH domains the only other known functional region is a C-terminal PDZ-binding motif that interacts with several PDZ binding proteins including the tyrosine-phosphatase-13 (PTPN13, previously known PTPL1 or FAP-1) [5] as well as the scaffolding proteins MUPP1 [3] and syntrophin [6].
One hypothesis that we had was TAPP1 and TAPP2 by specifically recognising PtdIns(3,4)P2 could function to recruit signalling molecules or complexes to the plasma membrane that down-regulate the PI 3-kinase signalling pathways [5]. Arguably, it makes sense to employ PtdIns(3,4)P2 as a signal to down-regulate the PI 3-kinase pathway, as the levels of this 3-phosphoinositide peaks later than PtdIns(3,4,5)P3. PtdIns(3,4)P2 would serve to down-regulate PI 3-kinase and signal the need for decreased formation of PtdIns(3,4,5)P3 production.
To test this idea Stephan Wullschleger generated knock-in mice expressing point mutants of TAPP1 and TAPP2 unable to interact with PtdIns(3,4)P2. The resulting TAPP1R211L/R211L TAPP2R218L/R218L double knock-in mice are viable and exhibited significantly enhanced activation of the Akt kinase a key downstream mediator of insulin signalling. By collaborating with David H. Wasserman at the Vanderbilt-NIH Mouse Metabolic Phenotyping Center, we were able to establish that double TAPP1/TAPP2 knock-in mice displayed significantly enhanced whole body insulin sensitivity in a hyperinsulinemic-euglycemic clamp study.
Stephan also demonstrated that TAPP1R211L/R211L TAPP2R218L/R218L double knock-in fibroblast cells that he derived displayed enhanced PtdIns(3,4,5)P3 levels and Akt activation in response to insulin. Overall Stephan's data suggests that enhanced insulin sensitivity is mediated by increased Akt kinase activation thereby stimulating glucose uptake in heart and skeletal muscle.
These results are important as the TAPP1R211L/R211L TAPP2R218L/R218L knock-in mice represent the first mouse model for these proteins and support the notion that TAPP1/TAPP2 operate as negative regulators of the PI 3-kinase signalling pathway. In future it will be exciting to work out what proteins TAPP1 and TAPP2 are interacting with and recruiting to the plasma membrane to dampen down the PI 3-kinase pathway. One idea we had was that it might be mediated by binding to PTPN13/PTPL1 but Stephan found that knock-in mice expressing catalytically inactive PTPN13C2374A/C2374A did not display enhanced insulin sensitivity.
To read a copy of Stephan's TAPP paper click (http://www.biochemj.org/bj/imps/abs/BJ20102012.htm)
References
1 Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G., Downes, C. P. and Alessi, D. R. (2000) Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem J. 351, 19-31
2 Thomas, C. C., Dowler, S., Deak, M., Alessi, D. R. and van Aalten, D. M. (2001) Crystal structure of the phosphatidylinositol 3,4-bisphosphate-binding pleckstrin homology (PH) domain of tandem PH-domain-containing protein 1 (TAPP1): molecular basis of lipid specificity. Biochem J. 358, 287-294
3 Kimber, W. A., Trinkle-Mulcahy, L., Cheung, P. C., Deak, M., Marsden, L. J., Kieloch, A., Watt, S., Javier, R. T., Gray, A., Downes, C. P., Lucocq, J. M. and Alessi, D. R. (2002) Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo. Biochem J. 361, 525-536
4 Marshall, A. J., Krahn, A. K., Ma, K., Duronio, V. and Hou, S. (2002) TAPP1 and TAPP2 are targets of phosphatidylinositol 3-kinase signaling in B cells: sustained plasma membrane recruitment triggered by the B-cell antigen receptor. Mol Cell Biol. 22, 5479-5491
5 Kimber, W. A., Deak, M., Prescott, A. R. and Alessi, D. R. (2003) Interaction of the protein tyrosine phosphatase PTPL1 with the PtdIns(3,4)P2-binding adaptor protein TAPP1. Biochem J. 376, 525-535
6 Hogan, A., Yakubchyk, Y., Chabot, J., Obagi, C., Daher, E., Maekawa, K. and Gee, S. H. (2004) The phosphoinositol 3,4-bisphosphate-binding protein TAPP1 interacts with syntrophins and regulates actin cytoskeletal organization. J Biol Chem. 279, 53717-53724
Christine Milburn (Dario Alessi's 3rd PhD Student, jointly supervised by Daan van Aalten) solved the structure of the C-terminal PH domain of TAPP1 revealing the mechanism by which it recognised PtdIns(3,4)P2 specifically [2]. Christine demonstrated that mutation of residues, Arg211 on TAPP1 or the equivalent Arg218 residue in TAPP2, that interacted with PtdIns(3,4)P2, completely prevented interaction of these proteins with PtdIns(3,4)P2 [2]. Interestingly, Christine structural data also suggested that binding of TAPP1 to PtdIns(3,4,5)P3 was inhibited by steric hindrance of an Ala residue located close to the position in which the 5'-phosphate would be expected to reside. The equivalent residue in PtdIns(3,4,5)P3-binding PH domains is frequently a Gly. Mutation of the Ala residue to Gly in TAPP1 resulted in it being capable of interacting with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity [2].
Wendy Kimber (Dario Alessi's 6th PhD Student) next provided the first evidence suggesting that TAPP1 binds PtdIns(3,4)P2 selectively in vivo, by showing that TAPP1 relocated from the cytosol to the plasma membrane of cells, following stimulation with agonists that induced PtdIns(3,4)P2, but not with those that induced mainly PtdIns(3,4,5)P3 [3]. Aaron Marshall's group in Winnipeg also demonstrated in B cells, both TAPP1 and TAPP2 translocated to the plasma membrane in response to antigen stimulation and this correlated with the formation of PtdIns(3,4)P2 rather than production of PtdIns(3,4,5)P3 [4].
Despite this work the biological functions of TAPP1 and TAPP2 remained elusive. Apart from the PH domains the only other known functional region is a C-terminal PDZ-binding motif that interacts with several PDZ binding proteins including the tyrosine-phosphatase-13 (PTPN13, previously known PTPL1 or FAP-1) [5] as well as the scaffolding proteins MUPP1 [3] and syntrophin [6].
One hypothesis that we had was TAPP1 and TAPP2 by specifically recognising PtdIns(3,4)P2 could function to recruit signalling molecules or complexes to the plasma membrane that down-regulate the PI 3-kinase signalling pathways [5]. Arguably, it makes sense to employ PtdIns(3,4)P2 as a signal to down-regulate the PI 3-kinase pathway, as the levels of this 3-phosphoinositide peaks later than PtdIns(3,4,5)P3. PtdIns(3,4)P2 would serve to down-regulate PI 3-kinase and signal the need for decreased formation of PtdIns(3,4,5)P3 production.
To test this idea Stephan Wullschleger generated knock-in mice expressing point mutants of TAPP1 and TAPP2 unable to interact with PtdIns(3,4)P2. The resulting TAPP1R211L/R211L TAPP2R218L/R218L double knock-in mice are viable and exhibited significantly enhanced activation of the Akt kinase a key downstream mediator of insulin signalling. By collaborating with David H. Wasserman at the Vanderbilt-NIH Mouse Metabolic Phenotyping Center, we were able to establish that double TAPP1/TAPP2 knock-in mice displayed significantly enhanced whole body insulin sensitivity in a hyperinsulinemic-euglycemic clamp study.
Stephan also demonstrated that TAPP1R211L/R211L TAPP2R218L/R218L double knock-in fibroblast cells that he derived displayed enhanced PtdIns(3,4,5)P3 levels and Akt activation in response to insulin. Overall Stephan's data suggests that enhanced insulin sensitivity is mediated by increased Akt kinase activation thereby stimulating glucose uptake in heart and skeletal muscle.
These results are important as the TAPP1R211L/R211L TAPP2R218L/R218L knock-in mice represent the first mouse model for these proteins and support the notion that TAPP1/TAPP2 operate as negative regulators of the PI 3-kinase signalling pathway. In future it will be exciting to work out what proteins TAPP1 and TAPP2 are interacting with and recruiting to the plasma membrane to dampen down the PI 3-kinase pathway. One idea we had was that it might be mediated by binding to PTPN13/PTPL1 but Stephan found that knock-in mice expressing catalytically inactive PTPN13C2374A/C2374A did not display enhanced insulin sensitivity.
To read a copy of Stephan's TAPP paper click (http://www.biochemj.org/bj/imps/abs/BJ20102012.htm)
References
1 Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G., Downes, C. P. and Alessi, D. R. (2000) Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem J. 351, 19-31
2 Thomas, C. C., Dowler, S., Deak, M., Alessi, D. R. and van Aalten, D. M. (2001) Crystal structure of the phosphatidylinositol 3,4-bisphosphate-binding pleckstrin homology (PH) domain of tandem PH-domain-containing protein 1 (TAPP1): molecular basis of lipid specificity. Biochem J. 358, 287-294
3 Kimber, W. A., Trinkle-Mulcahy, L., Cheung, P. C., Deak, M., Marsden, L. J., Kieloch, A., Watt, S., Javier, R. T., Gray, A., Downes, C. P., Lucocq, J. M. and Alessi, D. R. (2002) Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo. Biochem J. 361, 525-536
4 Marshall, A. J., Krahn, A. K., Ma, K., Duronio, V. and Hou, S. (2002) TAPP1 and TAPP2 are targets of phosphatidylinositol 3-kinase signaling in B cells: sustained plasma membrane recruitment triggered by the B-cell antigen receptor. Mol Cell Biol. 22, 5479-5491
5 Kimber, W. A., Deak, M., Prescott, A. R. and Alessi, D. R. (2003) Interaction of the protein tyrosine phosphatase PTPL1 with the PtdIns(3,4)P2-binding adaptor protein TAPP1. Biochem J. 376, 525-535
6 Hogan, A., Yakubchyk, Y., Chabot, J., Obagi, C., Daher, E., Maekawa, K. and Gee, S. H. (2004) The phosphoinositol 3,4-bisphosphate-binding protein TAPP1 interacts with syntrophins and regulates actin cytoskeletal organization. J Biol Chem. 279, 53717-53724