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Biomedical subjects

Louis Lim

Publications and source records attributed to Louis Lim.

25 records · Page 2Linked to original sources
PubMed2002

Characterization of RhoA-binding kinase ROKalpha implication of the pleckstrin homology domain in ROKalpha function using region-specific antibodies.

Rho-binding kinase alpha (ROKalpha) is a serine/threonine kinase with multiple functional domains involved in actomyosin assembly. It has previously been documented that the C terminus part of ROKalpha interacts with the N-terminal kinase domain and thereby regulates its catalytic activity. Here we used antibodies against different domains of ROKalpha and were able to reveal some structural aspects that are essential for the specific functions of ROKalpha. Antibodies against the kinase domain revealed that this part of the protein is highly complex and inaccessible. Further experiments confirmed that this domain could undergo inter- and intramolecular interactions in a complex manner, which regulates the kinase catalytic activity. Other antibodies that raised against the coiled-coil domain, Rho binding domain, and the pleckstrin homology (PH) domain were all effective in recognizing the native proteins in an immunoprecipitation assay. Only the anti-Rho binding domain antibodies could activate the kinase independent of RhoA. The PH antibodies had no apparent effects on the catalytic activity but were effective in blocking actomyosin assembly and cell contractility. Likewise, mutations of the PH domains can abrogate its dominant negative effects on actin morphology. The subsequent disruption of endogenous ROK localization to the actomyosin network by overexpressing the PH domain is supportive of a role of the PH domain of ROK in targeting the kinase to these structures.

Animals↗
PubMed2002

Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK).

BACKGROUND: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments. RESULTS: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization. CONCLUSION: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation.

Animals↗
PubMed2002

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.

ADP Ribose Transferases↗
PubMed2002

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.

3T3 Cells↗
PubMed2002

ACK family tyrosine kinase activity is a component of Dcdc42 signaling during dorsal closure in Drosophila melanogaster.

We have characterized Drosophila melanogaster ACK (DACK), one of two members of the ACK family of nonreceptor tyrosine kinases in Drosophila. The ACKs are likely effectors for the small GTPase Cdc42, but signaling by these proteins remains poorly defined. ACK family tyrosine kinase activity functions downstream of Drosophila Cdc42 during dorsal closure of the embryo, as overexpression of DACK can rescue the dorsal closure defects caused by dominant-negative Dcdc42. Similar to known participants in dorsal closure, DACK is enriched in the leading edge cells of the advancing epidermis, but it does not signal through activation of the Jun amino-terminal kinase cascade operating in these cells. Transcription of DACK is responsive to changes in Dcdc42 signaling specifically at the leading edge and in the amnioserosa, two tissues involved in dorsal closure. Unlike other members of the ACK family, DACK does not contain a conserved Cdc42-binding motif, and transcriptional regulation may be one route by which Dcdc42 can affect DACK function. Expression of wild-type and kinase-dead DACK transgenes in embryos, and in the developing wing and eye, reveals that ACK family tyrosine kinase activity is involved in a range of developmental events similar to that of Dcdc42.

Amino Acid Sequence↗