Thomas Leung

PAK Kinases Are Directly Coupled to the PIX Family of Nucleotide Exchange Factors

The PAK family of kinases are regulated through interaction with the small GTPases Cdc42 and Rac1, but little is known of the signaling components immediately upstream or downstream of these proteins. We have purified and cloned a new class of Rho-p21 guanine nucleotide exchange factor binding tightly through its N-terminal SH3 domain to a conserved proline-rich PAK sequence with a Kd of 24 nM. This PAK-interacting exchange factor (PIX), which is widely expressed and enriched in Cdc42- and Rac1-driven focal complexes, is required for PAK recruitment to these sites. PIX can induce membrane ruffling, with an associated activation of Rac1. Our results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex.

A Conserved Negative Regulatory Region in αPAK: Inhibition of PAK Kinases Reveals Their Morphological Roles Downstream of Cdc42 and Rac1

ABSTRACT αPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129–1143, 1997) resembling those of Cdc42G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in αPAK). αPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding αPAK83-149 potently inhibited GTPγS-bound Cdc42-mediated kinase activation of both αPAK and βPAK. Coexpression of this PAK inhibitor with Cdc42G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of αPAK83-149 completely blocked the phenotypic effects of hyperactive αPAKL107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.

RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

ABSTRACT The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.

p80 ROKα binding protein is a novel splice variant of CRMP-1 which associates with CRMP-2 and modulates RhoA-induced neuronal morphology

Using antibody against the Rho binding domain of ROKα, two neuronal phosphoproteins of 62 and 80 kDa were co‐immunoprecipitated from brain extracts. Peptide analysis revealed their identity as collapsin response mediator proteins (CRMPs); p62 was CRMP‐2 whereas p80 was a novel splice form of CRMP‐1 with an extended N‐terminus. p80 CRMP‐1 was able to complex with CRMP‐2, suggesting that p80 CRMP‐1 and CRMP‐2 form oligomers. CRMP‐2 was the major substrate of ROK. p80 CRMP‐1 interacted with the kinase domain of ROKα, resulting in inhibition of the catalytic activity towards other substrates. Over‐expression of p80 CRMP‐1 and CRMP‐2 together counteracted the effects of RhoA on neurite retraction, an effect enhanced by mutation of the ROK phosphorylation site in CRMP‐2. p80 CRMP‐1 and CRMP‐2 may be modulators of RhoA‐dependent signaling, through interaction with and regulation of ROKα.

Vimentin intermediate filament reorganization by Cdc42: involvement of PAK and p70 S6 kinase.

Rho family GTPases play a major role in actin cytoskeleton reorganization. Recent studies have shown that the activation of Rho family GTPases also induces collapse of the vimentin intermediate filament (IF) network in fibroblasts. Here, we report that Cdc42V12 induces the reorganization of vimentin IFs in Hela cells, and such reorganization is independent of actin and microtubule status. We analyzed the involvement of three serine/threonine kinase effectors, MRCK, PAK and p70 S6K in the Cdc42-induced vimentin reorganization. Surprisingly, the ROK-related MRCK is not involved in this IF reorganization. We detected phosphorylation of vimentin Ser72, a site phosphorylated by PAK, after Cdc42 activation. PAK inhibition partially blocked Cdc42-induced vimentin IF collapse suggesting the involvement of other effectors. We report that p70 S6 kinase (S6K)1 participates in this IF rearrangement since the inhibitor rapamycin or a dominant inhibitory S6K could reduce the Cdc42V12 or bradykinin-induced vimentin collapse. Further, inhibition of PAK and S6K in combination very effectively prevents Cdc42-induced vimentin IF collapse. Conversely, only in combination active PAK and S6K could induce a vimentin IF rearrangement that mimics the Cdc42 effect. Thus, Cdc42-induced vimentin reorganization involves PAK and, in a novel cytoskeletal role, p70 S6K.

Cdc42 antagonizes inductive action of cAMP on cell shape, via effects of the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) on myosin light chain phosphorylation

Rho GTPases play pivotal roles in regulating cell morphology. We previously showed that RhoA acts via ROKalpha to counteract the effects of the classical second messenger cyclic AMP on cell shape changes. Here we show that active Cdc42V12 also competes against the cAMP-induced stellate morphology in SH-EP cells. This Cdc42 effect is not mediated by the RhoA/ ROK pathway but rather the related MRCKalpha, a myotonic dystrophy kinase-related Cdc42-binding kinase. Co-expression of a dominant inhibitory MRCKalpha mutant with Cdc42V12 blocks the ability of the GTPase to counteract cAMP, suggesting that MRCK acts downstream of Cdc42 in this process. Cdc42V12 enhances the phosphorylation of myosin light chain (MLC) at the cell periphery and sustains focal adhesion complexes, while MLC kinase inhibitors destroy focal adhesion complexes and impair the Cdc42V12 protective effect. The data suggest that the maintenance of focal adhesion complexes via the regulation of myosin II activity underlies the ability of Cdc42 to protect against the effect of elevated cAMP.

Silencing the Metallothionein-2A Gene Induces Entosis in Adherent MCF-7 Breast Cancer Cells

The presence of a live cell cohabiting within another cell has fascinated scientists for many decades. Far from being a spurious event, many have attempted to uncover the molecular mechanism underlying this phenomenon. In this study, we observed anchorage‐dependent MCF‐7 cells internalizing neighboring epithelial cells (entosis) after siRNA‐mediated silencing of the Metallothionein‐2A (MT‐2A) gene. MTs belong to a family of low‐molecular weight proteins, which bind metal ions endogenously and its over‐expression has been reported in a variety of cancers that include breast, prostate, and colon. We provide microscopic evidence at light and ultrastructural levels of the occurrence of entosis after altering MT expression in a subpopulation of MCF‐7 breast cancer cells by silencing the MT‐2A gene. Our results demonstrate that adheren junctions may play important roles in the formation of cell‐in‐cell cytostructure after MT‐2A gene downregulation and the entotic process does not appear to involve genes associated with autophagy. Interiorized cells often underwent lysosomal degradation within the cytoplasmic body of the engulfing cell. It would appear that a subset of breast cancer cells could die via entosis after MT‐2A gene silencing. Anat Rec 293:1685–1691, 2010.

Genomic organization of human myotonic dystrophy kinase-related Cdc42-binding kinase α reveals multiple alternative splicing and functional diversity

Myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha) is a Cdc42/Rac interactive binding-containing serine/threonine kinase with multiple functional domains. Its roles in the regulation of peripheral actin reorganization in HeLa cells and NGF-induced neurite outgrowth in PC12 cells have been documented. Here we report the characterization of the genomic structure and alternative splicing of the human counterpart. Human MRCKalpha gene is located on chromosome 1q42.1, spanning a genomic region of 250-300 kb and is composed of 41 exons. Four exons in the internal variable region and six in the 3 end were found to undergo extensive alternative splicing, giving rise to 96 possible transcripts of different combinations. The region of the internal splice site that defines a variable region in between two functional domains of opposite regulatory effects on MRCKalpha catalytic activity, and the 3 end splice site that generates variants with differential GTPase binding activity suggest a role for these alternative splicing events in MRCKalpha regulation.