Ivan Tan

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 Tripartite Complex Containing MRCK Modulates Lamellar Actomyosin Retrograde Flow

Actomyosin retrograde flow underlies the contraction essential for cell motility. Retrograde flow in both lamellipodia and lamella is required for membrane protrusion and for force generation by coupling to cell adhesion. We report that the Rac/Cdc42-binding kinase MRCK and myosin II-related MYO18A linked by the adaptor protein LRAP35a form a functional tripartite complex, which is responsible for the assembly of lamellar actomyosin bundles and of a subnuclear actomyosin network. LRAP35a binds independently to MYO18A and MRCK. This binding leads to MRCK activation and its phosphorylation of MYO18A, independently of ROK and MLCK. The MRCK complex moves in concert with the retrograde flow of actomyosin bundles, with MRCK being able to influence other flow components such as MYO2A. The promotion of persistent protrusive activity and inhibition of cell motility by the respective expression of wild-type and dominant-negative mutant components of the MRCK complex show it to be crucial to cell protrusion and migration.

Characterization of RhoA-binding kinase ROK-alpha-implication of the PH domain in ROK-alpha function using region-specific antibodies

Rho-binding kinase α (ROKα) is a serine/threonine kinase with multiple functional domains involved in actomyosin assembly. It has previously been documented that the C terminus part of ROKα interacts with the N-terminal kinase domain and thereby regulates its catalytic activity. Here we used antibodies against different domains of ROKα and were able to reveal some structural aspects that are essential for the specific functions of ROKα. 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.

Intermolecular and intramolecular interactions regulate catalytic activity of myotonic dystrophy kinase-related Cdc42-binding kinase alpha.

ABSTRACT Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKα to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKα multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKα catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase–distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase 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.

Actin-binding and Cell Proliferation Activities of Angiomotin Family Members Are Regulated by Hippo Pathway-mediated Phosphorylation

Background: LATS kinase, one of the core kinases of Hippo pathway, phosphorylates and inactivates the downstream coactivator YAP/TAZ. Results: The angiomotin (Amot) family members are phosphorylated by LATS kinase. Conclusion: Phosphorylation of Amots by LATS kinase inhibits actin-binding, stabilizes Amot, and inhibits cell proliferation. Significance: Besides phosphorylating YAP/TAZ, LATS kinase may phosphorylate other components of the Hippo pathway. Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.

ACK Family Tyrosine Kinase Activity Is a Component of Dcdc42 Signaling during Dorsal Closure in Drosophila melanogaster

ABSTRACT 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.

Phosphorylation of Myosin Phosphatase Targeting Subunit 3 (MYPT3) and Regulation of Protein Phosphatase 1 by Protein Kinase A

Myosin phosphatase targeting subunit 3 (MYPT3) and transforming growth factor-β-inhibited membrane-associated protein (TIMAP) are two closely related myosin-binding targeting subunits of protein phosphatase 1 (PP1c) with a characteristic CAAX (where AA indicates aliphatic amino acid) box at the C termini. Here we show that MYPT3 can be a substrate for protein kinase A (PKA). We first mapped the multiple phosphorylation sites within a central conserved motif. Deletion or mutations of this motif resulted in enhancement of the associated PP1c activity, suggesting that phosphorylation of MYPT3 may play an important role in regulating PP1c catalytic activity. However, unlike the other known MYPTs, which upon phosphorylation inhibit PP1c, PKA phosphorylation of MYPT3 resulted in PP1c activation, indicating a different mode of action. There is a direct interaction between the central conserved phosphorylated site motif with the N-terminal ankyrin repeat region; this interaction was significantly reduced with MYPT3 phosphorylation or acidic phosphorylation site mutations, with concomitant alterations in biochemical and morphological consequences. We therefore propose a novel mechanism for the phosphorylation of MYPT3 by PKA and activation of the catalytic activity through direct interaction of a central region of MYPT3 with its N-terminal region.

Myosin light chain kinases Division of work in cell migration

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.