Kathleen A. Gallo

LRRK2 in Parkinson's disease : protein domains and functional insights

Parkinsons disease (PD) is the most common motor neurodegenerative disease. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have been linked recently with autosomal-dominant parkinsonism that is clinically indistinguishable from typical, idiopathic, late-onset PD. Thus, the protein LRRK2 has emerged as a promising therapeutic target for treatment of PD. LRRK2 is extraordinarily large and complex, with multiple enzymatic and protein-interaction domains, each of which is targeted by pathogenic mutations in familial PD. This review places the PD-associated mutations of LRRK2 in a structural and functional framework, with the ultimate aim of deciphering the molecular basis of LRRK2-associated pathogenesis. This, in turn, should advance our understanding and treatment of familial and idiopathic PD.

RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP.

RON, a cDNA homologous to the hepatocyte growth factor (HGF) receptor gene (MET), encodes a putative tyrosine kinase. Here we show that the RON gene is expressed in several epithelial tissues as well as in granulocytes and monocytes. The major RON transcript is translated into a glycosylated single chain precursor, cleaved into a 185 kDa heterodimer (p185RON) of 35 (alpha) and 150 kDa (beta) disulfide‐linked chains, before exposure at the cell surface. The Ron beta‐chain displays intrinsic tyrosine kinase activity in vitro, after immunoprecipitation by specific antibodies. In vivo, tyrosine phosphorylation of p185RON is induced by stimulation with macrophage stimulating protein (MSP), a protease‐like factor containing four ‘kringle’ domains, homologous to HGF. In epithelial cells, MSP‐induced tyrosine phosphorylation of p185RON is followed by DNA synthesis. p185RON is not activated by HGF, nor is the HGF receptor activated by MSP in biochemical and biological assays. p185RON is also activated by a pure recombinant protein containing only the N‐terminal two kringles of MSP. These data show that p185RON is a tyrosine kinase activated by MSP and that it is member of a family of growth factor receptors with distinct specificities for structurally related ligands.

Signalling: Mixed-lineage kinase control of JNK and p38 MAPK pathways

Mixed-lineage kinases (MLKs) are serine/threonine protein kinases that regulate signalling by the c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated-protein kinase (MAPK) pathways. MLKs are represented in the genomes of both Caenorhabditis elegans and Drosophila melanogaster. The Drosophila MLK Slipper regulates JNK to control dorsal closure during embryonic morphogenesis. In mammalian cells, MLKs are implicated in the control of apoptosis and are potential drug targets for many neurodegenerative diseases.

The Mixed Lineage Kinase SPRK Phosphorylates and Activates the Stress-activated Protein Kinase Activator, SEK-1

SPRK (also called PTK-1 and MLK-3), a member of the mixed lineage kinase subfamily of (Ser/Thr) protein kinases, encodes an amino-terminal SH3 domain followed by a kinase catalytic domain, two leucine zippers interrupted by a short spacer, a Rac/Cdc42 binding domain, and a long carboxyl-terminal proline-rich region. We report herein that SPRK activates the stress-activated protein kinases (SAPKs) but not ERK-1 during transient expression in COS cells; the p38 kinase is activated modestly (1.3-2 fold) but consistently. SPRK also activates cotransfected SEK-1/MKK-4, a dual specificity kinase which phosphorylates and activates SAPK. Reciprocally, expression of mutant, inactive SEK-1 inhibits completely the basal and SPRK-activated SAPK activity. Immunoprecipitated recombinant SPRK is able to phosphorylate and activate recombinant SEK-1 in vitro to an extent comparable to that achieved by MEK kinase-1. These results identify SPRK as a candidate upstream activator of the stress-activated protein kinases, acting through the phosphorylation and activation of SEK-1.

Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo. Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation

Src homology 3 domain (SH3)-containing proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the c-Jun NH2-terminal kinase pathway. The mechanisms by which SPRK activity is regulated are not well understood. The small Rho family GTPases, Rac and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including SPRK, which contain Cdc42/Rac interactive binding motifs. Coexpression of SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac interactive binding-like motif contains six of the eight consensus residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is required for SPRKs association with and activation by Cdc42. However, experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which Cdc42 increases SPRKs catalytic activity involves a change in thein vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase. These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.

MLK3 regulates paxillin phosphorylation in chemokine-mediated breast cancer cell migration and invasion to drive metastasis

MLK3 kinase activates multiple mitogen-activated protein kinases and plays a critical role in cancer cell migration and invasion. In the tumor microenvironment, prometastatic factors drive breast cancer invasion and metastasis, but their associated signaling pathways are not well-known. Here, we provide evidence that MLK3 is required for chemokine (CXCL12)-induced invasion of basal breast cancer cells. We found that MLK3 induced robust phosphorylation of the focal adhesion scaffold paxillin on Ser 178 and Tyr 118, which was blocked by silencing or inhibition of MLK3-JNK. Silencing or inhibition of MLK3, inhibition of JNK, or expression of paxillin S178A all led to enhanced Rho activity, indicating that the MLK3-JNK-paxillin axis limits Rho activity to promote focal adhesion turnover and migration. Consistent with this, MLK3 silencing increased focal adhesions and stress fibers in breast cancer cells. MLK3 silencing also decreased the formation of breast cancer lung metastases in vivo, and breast cancer cells derived from mouse lung metastases showed enhanced Ser 178 paxillin phosphorylation. Taken together, our findings suggest that the MLK3-JNK-paxillin signaling axis may represent a potential therapeutic target and/or prognostic marker in breast cancer metastasis.

Cdc42 Induces Activation Loop Phosphorylation and Membrane Targeting of Mixed Lineage Kinase 3

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation-dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.

MLK3 is critical for breast cancer cell migration and promotes a malignant phenotype in mammary epithelial cells

The malignant phenotype in breast cancer is driven by aberrant signal transduction pathways. Mixed-lineage kinase-3 (MLK3) is a mammalian mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK pathways. Depending on the cellular context, MLK3 has been implicated in apoptosis, proliferation, migration and differentiation. Here we investigated the effect of MLK3 and its signaling to MAPKs in the acquisition of malignancy in breast cancer. We show that MLK3 is highly expressed in breast cancer cells. We provide evidence that MLK3s catalytic activity and signaling to c-jun N-terminal kinase (JNK) is required for migration of highly invasive breast cancer cells and for MLK3-induced migration of mammary epithelial cells. Expression of active MLK3 is sufficient to induce the invasion of mammary epithelial cells, which requires AP-1 activity and is accompanied by the expression of several proteins corresponding to AP-1-regulated invasion genes. To assess MLK3s contribution to the breast cancer malignant phenotype in a more physiological setting, we implemented a strategy to inducibly express active MLK3 in the preformed acini of MCF10A cells grown in 3D Matrigel. Induction of MLK3 expression dramatically increases acinar size and modestly perturbs apicobasal polarity. Remarkably, MLK3 expression induces luminal repopulation and suppresses the expression of the pro-apoptotic protein BimEL, as has been observed in Her2/Neu-expressing acini. Taken together, our data show that MLK3–JNK–AP-1 signaling is critical for breast cancer cell migration and invasion. Our current study uncovers both a proliferative and novel antiapoptotic role for MLK3 in the acquisition of a malignant phenotype in mammary epithelial cells. Thus, MLK3 may be an important therapeutic target for the treatment of invasive breast cancer.

Dynamic Positive Feedback Phosphorylation of Mixed Lineage Kinase 3 by JNK Reversibly Regulates Its Distribution to Triton-soluble Domains

MLK3 (mixed lineage kinase 3) is a widely expressed, mammalian serine/threonine protein kinase that activates multiple MAPK pathways. Previously our laboratory used in vivo labeling/mass spectrometry to identify phosphorylation sites of activated MLK3. Seven of 11 identified sites correspond to the consensus motif for phosphorylation by proline-directed kinases. Based on these results, we hypothesized that JNK, or another proline-directed kinase, phosphorylates MLK3 as part of a feedback loop. Herein we provide evidence that MLK3 can be phosphorylated by JNK in vitro and in vivo. Blockade of JNK results in dephosphorylation of MLK3. The hypophosphorylated form of MLK3 is inactive and redistributes to a Triton-insoluble fraction. Recovery from JNK inhibition restores MLK3 solubility and activity, indicating that the redistribution process is reversible. This work describes a novel mode of regulation of MLK3, by which JNK-mediated feedback phosphorylation of MLK3 regulates its activation and deactivation states by cycling between Triton-soluble and Triton-insoluble forms.

Phosphorylation of golgin-160 by mixed lineage kinase 3

Golgin-160 is a member of the coiled-coil family of golgin proteins, which are proposed to regulate the structure of the Golgi complex. The C-terminal two-thirds of golgin-160 is predicted to form a coiled-coil domain and the N-terminal head domain contains several putative binding domains, regulatory motifs and phosphorylation sites. Recently, it has been demonstrated that caspase-dependent cleavage of the golgin-160 head domain occurs rapidly after induction of apoptosis. The role of golgin-160 phosphorylation and the functional implications for Golgi structure have not been defined. In this study, we investigated the kinase(s) responsible for phosphorylation of golgin-160. Signaling through the small G-protein Rac and mixed-lineage-kinase-3 (MLK3) resulted in increased phosphorylation of golgin-160. The intracellular distribution of MLK3 overlapped with that of golgin-160 and the two proteins could be co-immunoprecipitated. In vitro kinase assays demonstrated that MLK3 directly phosphorylates golgin-160 in the N-terminal head region between residues 96 and 259. Overexpression of MLK3 caused an enhanced caspase-dependent cleavage of golgin-160 at Asp139. Golgin-160 is the first non-kinase substrate of MLK3 identified, and phosphorylation by MLK3 might modulate cleavage of golgin-160 during apoptosis.