Noriko Yokoyama

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1: substrate specificity, autophosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.

Abundance, complexation, and trafficking of Wnt/β-catenin signaling elements in response to Wnt3a

Background Wnt3a regulates a canonical signaling pathway in early development that controls the nuclear accumulation of β-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes. Results Using totipotent mouse F9 teratocarcinoma cells expressing Frizzled-1 and biochemical analyses, we detail the influence of Wnt3a stimulation on the expression, complexation, and subcellular trafficking of key signaling elements of the canonical pathway, i.e., Dishevelled-2, Axin, glycogen synthase kinase-3β, and β-catenin. Cellular content of β-catenin and Axin, and phospho-glycogen synthase kinase-3β, but not Dishevelled-2, increases in response to Wnt3a. Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions. Dishevelled-2, in contrast, is found predominately in the cytosol, whereas β-catenin is localized to the plasma membrane-enriched fraction. Wnt3a stimulates trafficking of Dishevelled-2, Axin, and glycogen synthase kinase-3β initially to the plasma membrane, later to the nucleus. Bioluminescence resonance energy transfer measurements reveal that complexes of Axin with Dishevelled-2, with glycogen synthase kinase-3β, and with β-catenin are demonstrable and they remain relatively stable in response to Wnt3a stimulation, although trafficking has occurred. Mammalian Dishevelled-1 and Dishevelled-2 display similar patterns of trafficking in response to Wnt3a, whereas that of Dishevelled-3 differs from the other two. Conclusion This study provides a detailed biochemical analysis of signaling elements key to Wnt3a regulation of the canonical pathway. We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules. In contrast, we observe little effect of Wnt3a stimulation on the level of protein-protein interactions among these constituents of Axin-based complexes themselves.

Involvement of protein phosphatase 2A in the interleukin-3-stimulated Jak2-Stat5 signaling pathway.

In this study, we report that the tyrosine kinase, Janus kinase 2 (Jak2), associates with the serine/threonine protein phosphatase 2A (PP2A) in 32Dcl3 myeloid progenitor cells. The association between Jak2 and PP2A transiently increases following interleukin-3 (IL-3) stimulation and activation of Jak2. The catalytic subunit of PP2A is tyrosine phosphorylated by Jak2 in vitro and in vivo, resulting in inhibition of phosphatase activity. PP2A also associates with Stat5 in 32Dcl3 cells in an IL-3-dependent manner. Pretreatment of 32Dcl3 cells with okadaic acid (OA), an inhibitor of PP2A, resulted in increased tyrosine phosphorylation and nuclear translocation of Stat5. Our results suggest that PP2A plays a negative regulatory role in regulating the IL-3 signaling pathway via formation of complexes with Jak2 and Stat5.

Phosphorylation of WASP by the Cdc42-associated Kinase ACK1 DUAL HYDROXYAMINO ACID SPECIFICITY IN A TYROSINE KINASE

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.

Inhibition of Src by direct interaction with protein phosphatase 2A.

In this study, we report that Src kinase is inhibited by protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We carried out experiments in vitro using purified PP2A (AC dimer) and full‐length v‐Src or truncated forms of v‐Src. The inhibition of v‐Src by PP2A is concentration‐ and time‐dependent. Addition of okadaic acid, a PP2A phosphatase inhibitor, abolished the PP2A‐dependent inhibition of v‐Src. When experiments were carried out at 4°C under conditions where PP2A activity is inhibited, Src activity was unaffected by the presence of PP2A, suggesting that PP2A binding alone is insufficient to block Src activity. These results imply that PP2A activity is essential for inhibition of v‐Src. We also demonstrate that PP2A binds to the catalytic and the regulatory domains of v‐Src.

Protein phosphatase 2A interacts with the Src kinase substrate p130 CAS

In this study, we report that the Src substrate Cas (p130 Crk-associated substrate) associates with protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We investigated this interaction in cells expressing a temperature-sensitive mutant form of v-Src. v-Src activation (by shifting cells from the nonpermissive to the permissive temperature) led to an increase in the tyrosine phosphorylation of v-Src and Cas, as well as in the association between v-Src and Cas. v-Src has previously been shown to bind to PP2A and to phosphorylate the catalytic subunit of PP2A, resulting in inhibition of phosphatase activity. We found that the association between v-Src and PP2A decreased as cells were shifted to the permissive temperature. In contrast, the levels of PP2A that co-immunoprecipitated with Cas increased when v-Src was activated. We obtained similar results in pull-down experiments with immobilized Microcystin, a PP2A inhibitor. Serine/threonine phosphorylation of Cas has previously been shown to occur in a cell cycle regulated matter. Treatment of NIH3T3 cells with okadaic acid, a PP2A inhibitor, augments the serine/threonine phosphorylation of Cas that occurs at mitosis. Furthermore, PP2A dephosphorylates serine residues on Cas in vitro. Taken together, our results suggest that PP2A may be involved in the cell cycle-specific dephosphorylation of Cas.

Determinants for the interaction between Janus kinase 2 and protein phosphatase 2A

We have shown that the serine/threonine protein phosphatase 2A (PP2A) associates with the Jak2 tyrosine kinase in a myeloid progenitor line. In this study, we characterized the regions of Jak2 and PP2A responsible for association and evaluated the functional consequences of association. We demonstrate that PP2A interacts with truncated forms of Jak2 containing the JH1 catalytic domain. Using GST fusion proteins, we show that the isolated JH1 and JH3 domains of Jak2 bind directly to PP2A. Jak2 contains putative PP2A binding sequences (LXXLL) in the JH1 domain (residues 1078-1082) and in the JH3 domain (residues 474-478). Mutation of the LXXLL sequence in the JH1 domain decreased PP2A binding in vitro, while mutation of the similar JH3 sequence did not affect PP2A binding. We analyzed full-length Jak2 bearing the LXXLL mutation in Cos-7 cells for association with PP2A. The JH1 mutation impaired Jak2 activity and had a modest effect on PP2A binding. Finally, we show that a mutant form of the PP2A catalytic subunit lacking a site for phosphorylation (Y307F) binds more tightly to Jak2 than wild-type PP2A, consistent with a model where phosphorylation disrupts the Jak2-PP2A interaction.

Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling

Background Wnt3a stimulates cellular trafficking of key signaling elements (e.g., Axin, Dishevelled-2, β-catenin, and glycogen synthase kinase-3β) and primitive endoderm formation in mouse F9 embryonic teratocarcinoma cells. Results The role of phosphoprotein phosphatase-2A in signaling of the Wnt/β-catenin/Lef-Tcf-sensitive gene activation pathway was investigated. Wnt3a action attenuates phosphoprotein phosphatase-2A activity and stimulates the Lef/Tcf-sensitive gene transcription. Inhibiting phosphoprotein phosphatase-2A by okadaic acid, by treatment with siRNA (targeting the C-subunit of the enzyme), or by expression of SV40 small t antigen mimics Wnt3a action, increasing the cellular abundance of Axin and phospho-glycogen synthase kinase-3β as well as the trafficking of signaling elements in the Wnt/β-catenin pathway. Although mimicking effects of Wnt3a on the cellular abundance and trafficking of key signaling elements in the Wnt canonical pathway, suppression of phosphatase-2A alone did not provoke activation of the Lef/Tcf-sensitive transcriptional response, but did potentiate its activation by Wnt3a. Phosphoprotein phosphatase-2A and the scaffold phosphoprotein Dishevelled-2 display similarities in cellular trafficking in response to either Wnt3a or suppression of the phosphatase. A docking site for phosphoprotein phosphatase-2A in the DEP domain of Dishevelled-2 was identified. Conclusion In current study, we showed new roles of phosphoprotein phosphatase-2A in Wnt/β-catenin signaling pathway: effect on protein expression, effect on protein trafficking, retention of molecules in subcellular compartments, and regulation of enzymatic activity of several key players. Docking of phosphoprotein phosphatase-2A by Dishevelled-2 suppresses phosphatase activity and explains in part the central role of this phosphatase in the counterregulation of the Wnt/β-catenin signaling pathway.

IDENTIFICATION OF RESIDUES INVOLVED IN V-SRC SUBSTRATE RECOGNITION BY SITE-DIRECTED MUTAGENESIS

To study the role of the catalytic domain in v‐Src substrate specificity, we engineered three site‐directed mutants (Leu‐472 to Tyr or Trp and Thr‐429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild‐type v‐Src and the mutants using two series of peptides that varied at residues C‐terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate‐1 (IRS‐1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu‐472 or Thr‐429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C‐terminal to tyrosine). This was particularly evident in the case of the L‐472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu‐472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.

Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

Background Dishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/β-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called “signalsomes”, which propagate signals from the Wnt receptor Frizzled to downstream elements. Results Very large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a. Conclusion These data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.