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Protein kinase activity of phosphoinositide 3-kinase regulates β-adrenergic receptor endocytosis

Nature Cell Biology volume 7, pages 785–796 (2005)Cite this article

Abstract

Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent β-adrenergic receptor (βAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent βAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) compliments the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective βAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions of tropomyosin resulted in complete inhibition of βAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in βAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.

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Figure 1: Lipid and protein phosphorylation activity of PI(3)K mutants.
Figure 2: Protein and lipid phosphorylation activities of PI(3)K are required for β2AR internalization.
Figure 3: Lipid phosphorylation activity of PI(3)K is required for AP-2 recruitment to the receptor complex.
Figure 4: Protein kinase activity of PI(3)K phosphorylates a protein with Mr 30K–35K.
Figure 5: Non-muscle tropomyosin is a substrate for protein phosphorylation activity of PI(3)K.
Figure 6: βARK1-mediated endogenous PI(3)K activity at the membrane is required for non-muscle tropomyosin phosphorylation.
Figure 7: Endogenous non-muscle tropomyosin is required for β2AR internalization.
Figure 8: Phosphorylation-deficient tropomyosinS61A mutant blocks transferrin uptake.

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Acknowledgements

This work was supported in part by National Institutes of Health grants PO1 HL 075443 (to H.A.R) and Beginning grant-in-aid from AHA, Mid-Atlantic affiliate (to S.V.N.P.). A.J. received a Howard Hughes Medical Student Research Award. We thank W. Zou for her excellent technical assistance. We also thank M. Delahunty and X. Q. Zhang for excellent technical assistance with the SF9 cells. We would like to thank J. Richardson and D. Richardson (Department of Biochemistry, Duke University Medical Center) for advice and thoughtful insights on the crystallographic structure of PI(3)Kγ and the PI(3)K mutants.

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  1. Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, 27710, NC, USA

    Sathyamangla V. Naga Prasad, Arundathi Jayatilleke, Aasakiran Madamanchi & Howard A. Rockman

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Correspondence to Howard A. Rockman.

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Naga Prasad, S., Jayatilleke, A., Madamanchi, A. et al. Protein kinase activity of phosphoinositide 3-kinase regulates β-adrenergic receptor endocytosis. Nat Cell Biol 7, 785–796 (2005). https://doi.org/10.1038/ncb1278

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