Molecular Cloning and Sequencing of the Cytostatic G Protein-activated Protein Kinase PAK I

Jakobi, Rolf; Chen, Charng‐Jui; Tuazon, Polygena T.; Traugh, Jolinda A.

doi:10.1074/jbc.271.11.6206

Cited by 85 publications

(107 citation statements)

References 31 publications

Supporting

Mentioning

106

Contrasting

Order By: Relevance

“…The cDNA corresponding to the p21-binding domain (PBD) of p21 activated protein kinase PAK2 (amino acids 66-146) was amplified from PAK2 cDNA (18) by PCR and cloned between the NdeI and XbaI sites of the pGEX-2T vector. Glutathione S-transferase (GST)-Rhotekin-Rho binding domain (RBD) and GST-Raf1-Ras binding domain were generated as described previously (19,20).…”

Section: Methodsmentioning

confidence: 99%

RhoA Mediates Cyclooxygenase-2 Signaling to Disrupt the Formation of Adherens Junctions and Increase Cell Motility

et al. 2006

Self Cite

View full text Add to dashboard Cite

Cyclooxygenase-2 (COX-2) represents an important target for treatment and prevention of colorectal cancer. Although COX-2 signaling is implicated in promoting tumor cell growth and invasion, the molecular mechanisms that mediate these processes are largely unknown. In this study, we show that the RhoA pathway mediates COX-2 signaling to disrupt the formation of adherens junctions and increase cell motility. Disruption of adherens junctions promotes tumor cell invasion and metastasis and is often associated with tumor progression. We detected high levels of RhoA activity in HCA-7 colon carcinoma cells that constitutively express COX-2. Inhibition of COX-2 significantly reduced the levels of RhoA activity in HCA-7 cells, suggesting that constitutive expression of COX-2 stimulates RhoA activity. Interestingly, inhibition of COX-2 or silencing of COX-2 expression with small interfering RNA (siRNA) stimulated the formation of adherens junctions, concomitant with increased protein levels of E-cadherin and A-catenin. Furthermore, inhibition of RhoA or silencing of RhoA expression with siRNA increased the levels of E-cadherin and A-catenin. Inhibition of Rho kinases (ROCK), the RhoA effector proteins, also increased levels of E-cadherin and A-catenin and stimulated formation of adherens junctions. The motility of HCA-7 cells was significantly decreased when COX-2 or RhoA was inhibited. Therefore, our data reveal a novel molecular mechanism that links COX-2 signaling to disrupt the formation of adherens junctions; COX-2 stimulates the RhoA/ROCK pathway, which reduces levels of E-cadherin and A-catenin leading to disruption of adherens junction formation and increased motility. Understanding of COX-2 downstream signaling pathways that promote tumor progression is crucial for the development of novel therapeutic strategies. (Cancer Res 2006; 66(24): 11700-8)

show abstract

Section: Methodsmentioning

confidence: 99%

RhoA Mediates Cyclooxygenase-2 Signaling to Disrupt the Formation of Adherens Junctions and Increase Cell Motility

et al. 2006

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition to its effects on the actin cytoskeleton, PAKs may play a role in disassembling intermediate filaments composed of desmin (21). Studies on PAK as a protease-activated kinase (22) indicated that the catalytic domain needs to undergo ATP-dependent autotransphosphorylation prior to conversion to its active form (23). The behavior of PAK as a dimer in solution (9) suggests that transphosphorylation of the kinase can be rapid.…”

mentioning

confidence: 99%

The Mechanism of PAK Activation

Chong

Tan

Lim

et al. 2001

Journal of Biological Chemistry

224

View full text Add to dashboard Cite

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.

show abstract

“…The demonstration that a PAK regulates the activity of class I myosin provides a link to how members of the small GTPbinding proteins, like Rac, mediate some of their effects on the actin cytoskeleton and membrane ruffling of vertebrate cells in culture (7)(8)(9). Class I or other unconventional myosins, like the class VI myosins, may in turn be the functional connection between the regulatory activity of PAKs and the motor activity necessary for changes in the actin cytoskeleton and membrane ruffling.…”

mentioning

confidence: 99%

Constitutive Activation of Endocytosis by Mutation ofmyoA, the Myosin I Gene of Aspergillus nidulans

Yamashita

May

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

Class I myosins function in cell motility, intracellular vesicle trafficking and endocytosis. Recently, it was shown that class I myosins are phosphorylated by a member of the p21-activated kinase (PAK) family. PAK phosphorylates a conserved serine or threonine residue in the myosin heavy chain. Phosphorylation at this site is required for maximal activation of the actin-activated Mg 2؉ -ATPase activity in vitro. This serine or threonine residue is conserved in all known class I myosins of microbial origin and in the human and mouse class VI myosins. We have investigated the in vivo significance of this phosphorylation by mutating serine 371 of the class I myosin heavy chain gene myoA of Aspergillus nidulans. Mutation to glutamic acid, which mimics phosphorylation and therefore activation of the myosin, results in an accumulation of membranes in growing hyphae. This accumulation of membranes results from an activation of endocytosis. In contrast, mutation of serine 371 to alanine had no discernible effect on endocytosis. These studies are the first to demonstrate the in vivo significance of a regulatory phosphorylation on a class I myosin. Furthermore, our results suggest that MYOA has two functions, one dependent and one independent of phosphorylation.Class I myosins have been implicated in a variety of cellular processes, including cell locomotion, contractile vacuole function, receptor-mediated endocytosis, protein secretion, and intracellular vesicle transport (1). We have been studying the function of the class I myosin gene, myoA, of the filamentous fungus Aspergillus nidulans (2). We constructed a conditional null mutant myoA strain using the promoter of the inducible alcohol dehydrogenase gene alcA (2). Using this strain, we showed that myoA is an essential gene in A. nidulans and that it was required for the establishment of polarized hyphal growth. We also showed that strains lacking myoA were deficient for the secretion of acid phosphatase into the medium. In contrast, work on class I myosin function in the budding yeast Saccharomyces cerevisiae has suggested that they function in endocytosis and possibly protein secretion (3, 4). In S. cerevisiae there are two genes, MYO3 and MYO5, that code for class I myosin polypeptides. Deletion of either of these genes is not lethal, suggesting that they have overlapping or redundant functions. Deletion of both genes produces strains that either grow poorly (4) or are inviable (3). A strain that has a temperature-sensitive myo5 mutation is defective for endocytosis but not for protein secretion (3).Studies of class I myosin function and their actin-activated ATPase activity in Dictyostelium discoideum and Acanthamoeba castellanii have shown that these class I myosins are regulated by phosphorylation at a specific and highly conserved serine or threonine residue on the heavy chain (5, 6). This phosphorylation site is also found in other unconventional myosins like the class VI myosins from humans and mice (Fig. 1). Recent studies have shown that the kinase respon...

show abstract

Molecular Cloning and Sequencing of the Cytostatic G Protein-activated Protein Kinase PAK I

Cited by 85 publications

References 31 publications

RhoA Mediates Cyclooxygenase-2 Signaling to Disrupt the Formation of Adherens Junctions and Increase Cell Motility

RhoA Mediates Cyclooxygenase-2 Signaling to Disrupt the Formation of Adherens Junctions and Increase Cell Motility

The Mechanism of PAK Activation

Constitutive Activation of Endocytosis by Mutation ofmyoA, the Myosin I Gene of Aspergillus nidulans

Contact Info

Product

Resources

About