Jian P. Lian

Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks

ABSTRACT Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the α and βγ subunits of complex G proteins.

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

Neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) are known to exhibit rapid activation of four protein kinases with molecular masses of ~69, ~63, ~49, and ~40-kDa. Activation of these kinases is blocked by antagonists of phosphatidylinositol 3-kinase and type 1 and/or type 2A protein phosphatases. These enzymes can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297-331 of the 47-kDa subunit of the NADPH-oxidase complex fixed within a gel. In this report, we demonstrate that an antibody generated to a fusion protein containing amino acid residues 175-306 of p21-activated protein kinase 1 (Pak1) reacts with three proteins in guinea pig neutrophils with molecular masses in the 60-70-kDa range during Western blotting. This antibody immunoprecipitates both the 69- and 63-kDa renaturable kinases from lysates of stimulated cells along with a minor 60-kDa kinase. No activities were observed for any of these enzymes in immunoprecipitates from unstimulated neutrophils. However, addition of ATP and activated Rac 1 or Cdc42 to immunoprecipitates from unstimulated cells resulted in the stimulation of two renaturable kinases with molecular masses in the 69- and 63-kDa range. These immunoprecipitates also contained two novel protein kinases with masses of ~49 and 40 kDa that were selectively activated by Cdc42. In contrast, the 69- and 63-kDa kinases were not immunoprecipitated from lysates of stimulated neutrophils with an antibody to Pak2 or with nonimmune serum. These data indicate that the renaturable 69- and 63-kDa kinases are Paks and reveal some of the upstream events that are necessary for the rapid activation of this family of protein kinases in neutrophils.

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

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.

A Protein Kinase from Neutrophils That Specifically Recognizes Ser-3 in Cofilin

Cofilin promotes the depolymerization of actin filaments, which is required for a variety of cellular responses such as the formation of lamellipodia and chemotaxis. Phosphorylation of cofilin on serine residue 3 is known to block these activities. We now report that neutrophils contain a protein kinase that selectively catalyzes the phosphorylation of cofilin on serine 3 (≥70%) and a nonspecific kinase that recognizes multiple sites in this protein. The selective serine 3 cofilin kinase binds to a deoxyribonuclease I affinity column, whereas the nonspecific cofilin kinase does not. Deoxyribonuclease I forms a very tight complex with actin, and deoxyribonuclease affinity columns have been utilized to identify a variety of proteins that interact with the cytoskeleton. The serine 3 cofilin kinase did not react with antibodies to LIM kinase 1 or 2, which can catalyze the phosphorylation of cofilin in other cell types. The activity of the serine 3 cofilin kinase was insensitive to a variety of selective antagonists of protein kinases but was blocked by staurosporine. This pattern of inhibition is similar to that observed for the kinase that is active with cofilin in intact neutrophils. Thus, neutrophils contain a protein kinase distinct from LIM kinase-1/2 that selectively recognizes serine 3 in cofilin.

Phosphorylation of the Activation Loop of γ p21-Activated Kinase (γ-Pak) and Related Kinases (MSTs) in Normal and Stressed Neutrophils

Neutrophils stimulated with a variety of chemoattractants exhibit a rapid activation of two p21-activated kinases (Paks) with molecular masses of ∼63 and 69 kDa (γ- and α-Pak). A number of in vitro studies suggest that modification of Thr402 in the activation loop (AL) of γ-Pak can play a critical role in the regulation of this kinase under certain circumstances. A phosphospecific Ab was generated to this region of Pak (pPak(AL)Ab). This Ab reacted with activated γ- and α-Pak from fMLP-stimulated neutrophils that contain the sequence KRXT(P)XXGTP in their ALs. The rapid but transient activation of Paks in normal stimulated neutrophils coincided with phosphorylation and dephosphorylation at the ALs of these enzymes. In contrast, stressed cells exhibited a prolonged phosphorylation at Thr402 in both intact γ-Pak and a proteolytic fragment of this kinase. The pPak(AL)Ab also reacted with the mammalian sterile twenty-like kinases (MSTs) (members of the Pak family) in osmotically stressed neutrophils and neutrophils treated with certain apoptotic agents (i.e., tumor promoters that inhibit type 1 and 2A protein phosphatases) but not in normal fMLP-stimulated cells. Thus, our results indicate that the AL of γ-Pak undergoes transient phosphorylation during normal neutrophil stimulation and chronic phosphorylation in stressed cells. In addition, we demonstrate that a number of MSTs are present in neutrophils and also undergo phosphorylation during stressful circumstances.

Activation of the p21‐activated protein kinases from neutrophils with an antibody that reacts with the N‐terminal region of Pak 1

© 1997 Federation of European Biochemical Societies.

The P21-Activated Protein Kinases (Paks) Receive And Integrate Messages From A Variety of Signaling Pathways

Neutrophils stimulated with leukotriene B4, platelet activating factor or a variety of other chemoattractants that bind to G-protein coupled serpentine receptors exhibit rapid activation of two p21-activated kinases (Paks) with molecular masses of ca 63 and 69 kDa (y-and a- Pak)1.2.3 The 63 and 69 kDa Paks are ser/thr protein kinases that preferentially recognize the consensus sequence - (K/R)RX(S/T)- where X can be an acidic, basic or neutral amino acid4. These kinases can be conveniently assayed in neutrophils by their ability to undergo renaturation after separation by SDS/PAGE and undergoing autophosphorylation5 or catalyzing the phosphorylation of a peptide substrate fixed within a gel that corresponds to amino acid residues 297-331 of the 47 kDa subunit of the NADPH-oxidase complex (p47-phox) 1 .Histone H4 can also serve as a highly specific substrate for these enzymes in “in gel” assays5.

Functions of the P21-Activated Protein Kinases (Paks) in Neutrophils and their Regulation by Complex Lipids

Neutrophils stimulated with the chemoattractant fMet-Leu-Phe ( fMLP ) exhibit rapid activation of four renaturable protein kinases with molecular masses of ca. 69, 63, 49 and 40 kDa.1,2,3 The 69 and 63 kDa kinases were subsequently identified as p21-activated protein kinases ( Paks )4,5. These enzymes can be conveniently assayed by their ability to undergo renaturation after SDS/PAGE and catalyzing the phosphorylation of a peptide substrate fixed within a gel2,3 ( e.g., Fig. 1 ). The peptide substrate utilized corresponds to amino acid residues 297-331 of the 47 kDa subunit of the NADPH-oxidase complex ( p47-phox ).2,3 The preferred recognition/consensus sequence for Pak is - ( K/R )RXS — where X can be an acidic, basic or neutral amino acid.6 There are three sites in the p47-phox peptide which contain this sequence (ser-304, -320 &-328 ).