Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3-phosphoinositide-dependent kinase 1.
Protein kinase N 1 (PKN1), which in part resembles yeast protein kinase C, has been shown to be under the control of Rho GTPases and 3-phosphoinositide-dependent kinase 1 (PDK1). We show here that green fluorescent protein-tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation, and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation is dependent on Rac1 activation, although the GTPase binding HR1abc domain is not sufficient for this recruitment. The PKN1 kinase domain, however, localizes constitutively to this compartment, and we demonstrate that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1-positive compartment upon hyperosmotic stress. Taken together, our findings present a pathway for the selective hyperosmotic-induced Rac1-dependent PKN1 translocation and PDK1-dependent activation.
Torbett
Neil E
NE
Protein Phosphorylation Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Casamassima
Adele
A
Parker
Peter J
PJ
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D009995:Osmosis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019869:Phosphatidylinositol 3-Kinases; C061570:protein kinase N; D011493:Protein Kinase C; D020830:rac1 GTP-Binding Protein; C049347:fasudil