Browse
Submit Data
Databases
API
Help

Dataset Information

38 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Recognition of physiological phosphorylation sites by p21-activated kinase 4.

ABSTRACT: Many serine/threonine protein kinases discriminate between serine and threonine substrates as a filter to control signaling output. Among these, the p21-activated kinase (PAK) group strongly favors phosphorylation of Ser over Thr residues. PAK4, a group II PAK, almost exclusively phosphorylates its substrates on serine residues. The only well documented exception is LIM domain kinase 1 (LIMK1), which is phosphorylated on an activation loop threonine (Thr508) to promote its catalytic activity. To understand the molecular and kinetic basis for PAK4 substrate selectivity we compared its mode of recognition of LIMK1 (Thr508) with that of a known serine substrate, β-catenin (Ser675). We determined X-ray crystal structures of PAK4 in complex with synthetic peptides corresponding to its phosphorylation sites in LIMK1 and β-catenin to 1.9 Å and 2.2 Å resolution, respectively. We found that the PAK4 DFG + 1 residue, a key determinant of phosphoacceptor preference, adopts a sub-optimal orientation when bound to LIMK1 compared to β-catenin. In peptide kinase activity assays, we find that phosphoacceptor identity impacts catalytic efficiency but does not affect the K_m value for both phosphorylation sites. Although catalytic efficiency of wild-type LIMK1 and β-catenin are equivalent, T508S mutation of LIMK1 creates a highly efficient substrate. These results suggest suboptimal phosphorylation of LIMK1 as a mechanism for controlling the dynamics of substrate phosphorylation by PAK4.

SUBMITTER: Chetty AK

PROVIDER: S-EPMC7395882 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Protein kinase D-mediated phosphorylation at Ser99 regulates localization of p21-activated kinase 4.

Project description:PAKs (p21-activated kinases) are effectors of RhoGTPases. PAK4 contributes to regulation of cofilin at the leading edge of migrating cells through activation of LIMK (Lin-11/Isl-1/Mec-3 kinase). PAK4 activity is regulated by an autoinhibitory domain that is released upon RhoGTPase binding as well as phosphorylation at Ser474 in the activation loop of the kinase domain. In the present study, we add another level of complexity to PAK4 regulation by showing that phosphorylation at Ser99 is required for its targeting to the leading edge. This phosphorylation is mediated by PKD1 (protein kinase D1). Phosphorylation of PAK4 at Ser99 also mediates binding to 14-3-3 protein, and is required for the formation of a PAK4-LIMK-PKD1 complex that regulates cofilin activity and directed cell migration.

| S-EPMC3880571 | biostudies-literature

LKB1 suppresses p21-activated kinase-1 (PAK1) by phosphorylation of Thr109 in the p21-binding domain.

Project description:The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr(109) in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr(109) by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/-) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.

| S-EPMC2881753 | biostudies-literature

Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

Project description:Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.

| S-EPMC7568686 | biostudies-literature

Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

Project description:Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

| S-EPMC3543003 | biostudies-literature

Protein kinase D1 (PKD1) phosphorylation on Ser<sup>203</sup> by type I p21-activated kinase (PAK) regulates PKD1 localization.

Project description:Although PKC-mediated phosphorylation of protein kinase D1 (PKD1) has been extensively characterized, little is known about PKD1 regulation by other upstream kinases. Here we report that stimulation of epithelial or fibroblastic cells with G protein-coupled receptor agonists, including angiotensin II or bombesin, induced rapid and persistent PKD1 phosphorylation at Ser203, a highly conserved residue located within the PKD1 N-terminal domain. Exposure to PKD or PKC family inhibitors did not prevent PKD1 phosphorylation at Ser203, indicating that it is not mediated by autophosphorylation. In contrast, several lines of evidence indicated that the phosphorylation of PKD1 at Ser203 is mediated by kinases of the class I PAK subfamily, specifically 1) exposing cells to four structurally unrelated PAK inhibitors (PF-3758309, FRAX486, FRAX597, and IPA-3) that act via different mechanisms abrogated PKD1 phosphorylation at Ser203, 2) siRNA-mediated knockdown of PAK1 and PAK2 in IEC-18 and Swiss 3T3 cells blunted PKD1 phosphorylation at Ser203, 3) phosphorylation of Ser203 markedly increased in vitro when recombinant PKD1 was incubated with either PAK1 or PAK2 in the presence of ATP. PAK inhibitors did not interfere with G protein-coupled receptor activation-induced rapid translocation of PKD1 to the plasma membrane but strikingly prevented the dissociation of PKD1 from the plasma membrane and blunted the phosphorylation of nuclear targets, including class IIa histone deacetylases. We conclude that PAK-mediated phosphorylation of PKD1 at Ser203 triggers its membrane dissociation and subsequent entry into the nucleus, thereby regulating the phosphorylation of PKD1 nuclear targets, including class IIa histone deacetylases.

| S-EPMC5465480 | biostudies-literature

Protein kinase CK2 phosphorylates and activates p21-activated kinase 1.

Project description:Activation of the p21-activated kinase 1 (PAK1) is achieved through a conformational change that converts an inactive PAK1 dimer to an active monomer. In this paper, we show that this change is necessary but not sufficient to activate PAK1 and that it is, rather, required for CK2-dependent PAK1(S223) phosphorylation that converts a monomeric PAK1 into a catalytically active form. This phosphorylation appears to be essential for autophosphorylation at specific residues and overall activity of PAK1. A phosphomimetic mutation (S223E) bypasses the requirement for GTPases in PAK1 activation, whereas the constitutive activity of the PAK1 mutant (PAK1(H83,86L)), postulated to mimic GTPase-induced structural changes, is abolished by inhibition of S223 phosphorylation. Thus, S223 is likely accessible to CK2 upon conformational changes of PAK1 induced by GTPase-dependent and GTPase-independent stimuli, suggesting that S223 phosphorylation may play a key role in the final step of the PAK1 activation process. The physiological significance of this phosphorylation is reinforced by the observations that CK2 is responsible for epidermal growth factor-induced PAK1 activation and that inhibition of S223 phosphorylation abrogates PAK1-mediated malignant transformation of prostate epithelial cells. Taken together, these findings identify CK2 as an upstream activating kinase of PAK1, providing a novel mechanism for PAK1 activation.

| S-EPMC3771959 | biostudies-literature

GIT1 activates p21-activated kinase through a mechanism independent of p21 binding.

Project description:p21-activated kinases (PAKs) associate with a guanine nucleotide exchange factor, Pak-interacting exchange factor (PIX), which in turn binds the paxillin-associated adaptor GIT1 that targets the complex to focal adhesions. Here, a detailed structure-function analysis of GIT1 reveals how this multidomain adaptor also participates in activation of PAK. Kinase activation does not occur via Cdc42 or Rac1 GTPase binding to PAK. The ability of GIT1 to stimulate alphaPAK autophosphorylation requires the participation of the GIT N-terminal Arf-GAP domain but not Arf-GAP activity and involves phosphorylation of PAK at residues common to Cdc42-mediated activation. Thus, the activation of PAK at adhesion complexes involves a complex interplay between the kinase, Rho GTPases and protein partners that provide localization cues.

| S-EPMC387736 | biostudies-literature

p21-Activated Kinase 1 Promotes Breast Tumorigenesis <i>via</i> Phosphorylation and Activation of the Calcium/Calmodulin-Dependent Protein Kinase II.

Project description:p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.

| S-EPMC8802317 | biostudies-literature

Protein kinase D regulates cofilin activity through p21-activated kinase 4.

Project description:Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.

| S-EPMC3190831 | biostudies-literature

Phosphoinositides are essential coactivators for p21-activated kinase 1.

Project description:Phospholipid-enriched membranes such as the plasma membrane can serve as direct regulators of kinase signaling. Pak1 is involved in growth factor signaling at the plasma membrane, and its dysregulation is implicated in cancer. Pak1 adopts an autoinhibited conformation that is relieved upon binding to membrane-bound Rho GTPases Rac1 or Cdc42, but whether lipids also regulate Pak1 in vivo is unknown. We show here that phosphoinositides, particularly PIP(2), potentiate Rho-GTPase-mediated Pak1 activity. A positively charged region of Pak1 binds to phosphoinositide-containing membranes, and this interaction is essential for membrane recruitment and activation of Pak1 in response to extracellular signals. Our results highlight an active role for lipids as allosteric regulators of Pak1 and suggest that Pak1 is a "coincidence detector" whose activation depends on GTPases present in phosphoinositide-rich membranes. These findings expand the role of phosphoinositides in kinase signaling and suggest how altered phosphoinositide metabolism may upregulate Pak1 activity in cancer cells.

| S-EPMC3026281 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data