The Renaturable 69- and 63-kDa Protein Kinases That Undergo Rapid Activation in Chemoattractant-stimulated Guinea Pig Neutrophils Are p21-Activated Kinases

Ding, Jiabing; Knaus, Ulla G.; Lian, Jian; Bokoch, Gary M.; Badwey, John A.

doi:10.1074/jbc.271.40.24869

Cited by 51 publications

(62 citation statements)

References 43 publications

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“…Our observation of PAK1 staining in ruffles and lamellipodia at as early as 30 s after fMLP addition suggests that PAK1 relocalization is an early response to the activation of neutrophils by chemoattractant. These kinetics agree with biochemical studies which show that fMLP activates PAK by 15-30 s after stimulation [25,39]. The enhancement of PAK1 staining in the leading edge of motile neutrophils at later times, i.e.…”

Section: Discussionsupporting

confidence: 80%

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils

Dharmawardhane

Brownson

Lennartz

et al. 1999

Journal of Leukocyte Biology

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Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases. Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited. The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts. PAK1 colocalizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements. We investigated whether PAK1 was associated with remodeling of the actin cytoskeleton in activated human neutrophils. We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ). PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells. Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process. Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization. Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin. Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils. J. Leukoc. Biol. 66: 521-527; 1999.

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Section: Discussionsupporting

confidence: 80%

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils

Dharmawardhane

Brownson

Lennartz

et al. 1999

Journal of Leukocyte Biology

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“…5 and 6) is consistent with a similar stimulatory pathway being present in these cells. Addition of a GEF to this pathway, which is synergistically activated by PI 3-K and tyrosine phosphorylation as described above, will account for all of the data presented herein and in earlier publications (2)(3)(4)(5)(12)(13)(14). This modified model predicts that the Ca 2ϩ /CaM complex is required for activation of a tyrosine kinase that lies upstream of the GEF for Rac.…”

Section: Discussionmentioning

confidence: 79%

“…These protein kinases include two p21-activated protein kinases (Paks) 3 with molecular masses of 63 and 69 kDa (␥-and ␣-Pak; Refs. [1][2][3][4][5] and certain mitogen-activated protein kinase (MAPK) cascades (e.g., extracellular signal-regulated kinases (ERK-1/2), p38-MAPK; Refs. 6 -10).…”

mentioning

confidence: 99%

Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils

Lian

Crossley

Zhan

et al. 2001

The Journal of Immunology

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Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of ∼63 and 69 kDa (γ- and α-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), the intracellular Ca2+ channel (8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC50 values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N-(6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca2+/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.

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“…This technique has been used in the past to examine activation of Pak in activated neutrophils. 23 Figure 2e shows that in 3 of 4 control tumor pairs there is an enhanced activation of a 65 kDa band. When the bands for the whole group were analyzed by densitometry (Fig.…”

Section: Resultsmentioning

confidence: 93%

Dysregulation of phosphatidylinositol 3‐kinase and downstream effectors in human breast cancer

Salh

Marotta

Wagey

et al. 2001

Intl Journal of Cancer

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Phosphatidylinositol 3-kinase (PI3-K) is a growth factoractivated transforming lipid (and protein) kinase, involved in cell motility and invasion, that has multiple effectors. Relatively little is known about its expression and enzymatic activity in human breast cancer. Since growth factor receptors are amplified in breast cancer, and the tumor suppressor PTEN may be mutated in human breast cancer, it was hypothesized that PI3-K and its downstream effectors would be activated in this disease. In 11 resected tumors analyzed for expression of this kinase, a mean 3-fold increase in protein expression was observed over the corresponding adjacent control tissue. Using an in vitro lipid kinase assay of the immunoprecipitated PI3-K protein, a greater than 2-fold increase in activation was observed. These changes were observed in the absence of an activation of either protein kinase B (PKB, akt1) or p70 S6 kinase (p70 S6K). However, p21-activated kinase (Pak), p38 mitogen-activated protein kinase (p38 MAPK) and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK 2) were all overexpressed and demonstrated increased enzyme activity. It may be concluded that aberrant mitogenic signaling in human breast cancer in vivo involves Pak, p38 MAPK and MAPKAPK2 downstream of PI3-K, but neither of PKB or p70 S6K. It is proposed that this pathway may serve as a useful targeting nexus for investigation of small molecule inhibitors in human breast cancer.

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The Renaturable 69- and 63-kDa Protein Kinases That Undergo Rapid Activation in Chemoattractant-stimulated Guinea Pig Neutrophils Are p21-Activated Kinases

Cited by 51 publications

References 43 publications

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils

Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils

Dysregulation of phosphatidylinositol 3‐kinase and downstream effectors in human breast cancer

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