Multisite Autophosphorylation of p21-activated Protein Kinase γ-PAK as a Function of Activation

219

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both ␣-and ␤PAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK Nterminal regulatory domain play no direct role in activation, a phosphorylation of ␣PAK serine 144 or ␤PAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.

Section: Conserved Autophosphorylation Sites Among Different Pakmentioning

confidence: 96%

Section: Pak Autophosphorylation and Regulation Of Kinase Function-bementioning

confidence: 99%

See 1 more Smart Citation

The Mechanism of PAK Activation

Chong

Tan

Lim

et al. 2001

219

“…First, NAK primary phosphorylates serine residues, as PAK2 (83). Second, following anti-Nef or anti-PAK2 immunoprecipitations, the substrates of the IVKA not only show a similar pattern of phosphorylation but also have a similar phospho-peptide fingerprint.…”

Section: Discussionmentioning

confidence: 99%

Activation of p21-activated Kinase 2 and Its Association with Nef Are Conserved in Murine Cells but Are Not Sufficient to Induce an AIDS-like Disease in CD4C/HIV Transgenic Mice

Vincent

Priceputu

Kay

et al. 2006

A well conserved feature of human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus (SIV) Nef is the interaction with and activation of the human p21-activated kinase 2 (PAK2). The conservation of this interaction in other species and its significance for Nef pathogenesis in vivo are poorly documented. In the present study, we measured these parameters in Nef-expressing thymocytes, macrophages, and dendritic cells of a transgenic (Tg) mouse model of AIDS (CD4C/HIV). We found that Nef binds to and activates PAK2, but not PAK1 and -3, in these three cell subsets. Nef associates with only a small fraction of PAK2. The Nef-PAK2 complex also comprises ␤-PIX-COOL. The impact of the Nef-PAK2 association on disease development was also analyzed in Tg mice expressing 10 different Nef mutant alleles. CD4C/HIV Tg mice expressing Nef alleles defective in Nef-PAK2 association (P69A, P72A/P75A, R105A/R106A, ⌬56 -66, or G2A (myristoylation site)) failed to develop disease of the non-lymphoid organs (kidneys and lungs). Among these, only Tg mice expressing Nef P69A and Nef G2Ashowed some depletion of CD4 ؉ T cells, although a down-regulation of the CD4 surface protein was documented in all these Tg lines, except those expressing Nef ⌬56 -66 . Among other Tg mice expressing Nef mutants having conserved the Nef-PAK2 association (RD35AA, D174K, P147A/P150A, ⌬8 -17, and ⌬25-65), only Tg mice expressing Nef ⌬8 -17 develop kidney and lung diseases, but all showed partial CD4 ؉ T cell depletion despite some being defective for CD4 down-regulation (RD35AA and D174K). Therefore, Nef can activate murine PAK2 and associate with a small fraction of it, as in human cells. Such activation and binding of PAK2 is clearly not sufficient but may be required to induce a multiorgan AIDS-like disease in Tg mice.

“…Active Rac GTP precipitated by PAK-PBD was separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted for pan-Rac, followed by ECL detection. Left lanes, the total signal detected using cytosol preloaded with Chemokine-induced PI3K␥-Rac-Pak Signaling 43276 loop of the kinase domain and is known to be required for activation (41)(42)(43). The phospho-PAK Thr antibody demonstrated that RANTES stimulation of macrophages for 10 and 30 s triggered phosphorylation of PAK2 Thr-402 , but less so in PI3K␥Ϫ/Ϫ BMDM (Fig.…”

Section: Rantes-induced Rac Activationmentioning

confidence: 99%

Involvement of Phosphoinositide 3-Kinase γ, Rac, and PAK Signaling in Chemokine-induced Macrophage Migration

Weiss-Haljiti

Pasquali

et al. 2004

In macrophages, chemotactic stimuli cause the activation of Rac and PAK, but little is known about the signaling pathways involved and their role in chemotactic gradient sensing. Herein, we report that in macrophages, the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 activates the small GTPase Rac and its downstream target PAK2 within seconds. This response depends on G i activation and largely on the subsequent triggering of phosphoinositide 3-kinase ␥ (PI3K␥) and Rac. Retroviral transduction of tagged Rac1 and -2 indicates that RAN-TES/CCL5-mediated activation of PI3K␥ triggers Rac1 but not Rac2. In agreement, silencing of Rac1 by shRNA blocks PAK2 activity and inhibits RANTES/CCL5-induced macrophage polarization and directional migration. On the other hand, the tyrosine kinase receptor agonist CSF-1 activates PAK2 independently of PI3K␥ and Rac. Our results thus demonstrate a chemokinespecific signaling pathway in which G i and PI3K␥ coordinate to drive Rac1 and PAK2 activation that eventually controls the chemotactic response.Rac1, Rac2, and Rac3 constitute a subfamily of the Rho family of monomeric GTPases and cycle between active GTP-bound (Rac GTP ) and inactive GDP-bound (Rac GDP ) states (1-3). Activation is accomplished by guanine-nucleotide exchange factors (GEFs), 1 which catalyze GDP dissociation (4), and inactivation by GTPase-activating proteins, which increase the intrinsic GTPase activity (5). Rho GTPases integrate signals from cellular receptors and membrane components to regulate the cytoskeleton dynamics required for cell locomotion during chemotaxis, phagocytosis, and many other cellular responses (2, 6).Leukocyte chemotaxis toward sites of inflammation (7) is primarily mediated by chemokine signaling. RANTES CCL5 is a disease-relevant and potent chemoattractant for macrophages. Moreover, it has been shown that RANTES-mediated T-cell activation and chemotaxis requires Rho GTPase activity (8,9). RANTES is a member of the CC-subfamily of chemokines and activates the seven-transmembrane CC-chemokine receptors CCR1, CCR3, CCR4, and CCR5, which are coupled to pertussis toxin sensitive heterotrimeric G i␣ proteins (10). PI3K was identified as a target of these G-protein-coupled chemokine receptors because chemotaxis and polarization of T cells could be inhibited by isoform non-selective class I PI3K inhibitors, such as LY294002 (11). All class I PI3Ks are heterodimers consisting of a 110-kDa (p110) catalytic subunit and an 85-(p85) or 101-kDa (p101) regulatory subunit (12). The two catalytic subunits p110␣ and -␤ are ubiquitously expressed, whereas p110␥ and -␦ expression is largely confined to leukocytes. In addition to their characteristic expression, PI3Ks are differentially regulated by GPCRs and receptor tyrosine kinases (RTKs) (13). RTKs have been shown to activate p110␣, -␤, and -␦ via the p85 regulatory subunit (class I A: PI3K-␣, -␤, and -␦). In contrast, it has been shown that regulation of p110␥ (class I B: PI3K␥) is mediated via the p101 adapter, ...