Tonic protein kinase A activity maintains inactive beta2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase.
Activation of beta2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase beta2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of beta2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on beta2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated beta2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of beta2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of beta2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.
Chilcoat
Clayton D
CD
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA.
Sharief
Yousuf
Y
Jones
Samuel L
SL
D000328:Adult; D018821:CD18 Antigens / Q000097:blood* / Q000187:drug effects; D002227:Carbazoles / Q000494:pharmacology; D002448:Cell Adhesion; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D006706:Homeostasis; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D009219:Myosin-Light-Chain Kinase / Q000097:blood*; D009504:Neutrophils / Q000201:enzymology / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011758:Pyrroles / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000097:blood*
D018821:CD18 Antigens; D002227:Carbazoles; D007211:Indoles; D009842:Oligopeptides; D011758:Pyrroles; C057416:KT 5720; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase