Cooperative regulation of p70S6 kinase by receptor tyrosine kinases and G protein-coupled receptors augments airway smooth muscle growth.
We have previously demonstrated that concomitant activation of receptor tyrosine kinases and certain G protein-coupled receptors (GPCRs) can promote a synergistic increase in the rate of airway smooth muscle cell (ASM) proliferation. Here we clarify the role of p70S6 kinase (p70S6K) as an integrator of receptor tyrosine kinase and GPCR signaling that augments ASM DNA synthesis by demonstrating that specific p70S6K phosphorylation sites receive distinct regulatory input from GPCRs that promotes sustained kinase activity critical to mitogenesis. Prolonged stimulation of ASM cells with EGF and thrombin induced a greater than additive effect in levels of p70S6K phosphorylated at residue T389, whereas a significant but more modest increase in the level of T229 and T421/S424 phosphorylation was also observed. The augmenting effects of thrombin could be dissociated from p42/p44 MAPK activation, as selective inhibition of thrombin-stimulated p42/p44 failed to alter the profile of cooperative p70S6K T389 phosphorylation, p70S6K kinase activity, or ASM [(3)H]thymidine incorporation. Thrombin stimulated a sustained increase in the level of Akt phosphorylation and also augmented EGF-stimulated Akt phosphorylation. The cooperative effects of thrombin on Akt/p70S6K phosphorylation and [(3)H]thymidine incorporation were all attenuated by heterologous expression of Gbetagamma sequestrants. These data suggest that PI3K-dependent T389/T229 phosphorylation is limiting in late-phase p70S6K activation by EGF and contributes to the cooperative effect of GPCRs on p70S6K activity and cell growth.
Billington
Charlotte K
CK
Department of Internal Medicine, Wake Forest University Health Sciences Center, Winston-Salem, North Carolina 27157, USA.
Kong
Kok C
KC
Bhattacharyya
Raja
R
Wedegaertner
Philip B
PB
Panettieri
Reynold A
RA
Chan
Tung O
TO
Penn
Raymond B
RB
D001980:Bronchi / Q000033:anatomy & histology*; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2; D006801:Humans; D008938:Mitosis / Q000502:physiology; D009130:Muscle, Smooth / Q000502:physiology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013917:Thrombin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases / Q000235:genetics / Q000378:metabolism
D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D013917:Thrombin