The Scaffold Protein c-Jun NH2-Terminal Kinase-associated Leucine Zipper Protein Regulates Cell Migration through Interaction with the G Protein G 13

Abstract

Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH 2 -terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of fulllength shRNA-resistant mouse JLP rescued the impaired cell migration of the JLPdepleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G a13 , showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G a13 enhanced the migration of control HeLa cells, the G a13 -induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with G a13 .Key words: cell migration, MAP kinase, p38, RNA interference, scaffold protein.Abbreviations: DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; GST, glutathione S-transferase; HEK, human embryonic kidney; JLP, JNK-associated leucine zipper protein; JNK, c-Jun NH2-terminal kinase; JSAP1, JNK/stress-activated protein kinase-associated protein 1; KD, knockdown; MAPK, MAP kinase; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; shRNA, short hairpin RNA.MAP kinase (MAPK) cascades transmit signals through the sequential phosphorylation and activation of threetier kinase modules, consisting of MAPK kinase kinase, MAPK kinase and MAPK. Mammalian MAPK signalling pathways play a key role in multiple cellular functions, such as proliferation, migration and apoptosis (1, 2). The specificity of MAPK signalling is mediated, at least in part, by scaffold proteins. Scaffold proteins for MAPK cascades organize the MAPK signalling components into functional modules, thereby enabling the efficient activation of specific MAPK pathways (3-5). The scaffolding complexes also protect the signalling components within the relevant MAPK modules from undesired activation by other signalling molecules in cells.c-Jun NH 2 -terminal kinase (JNK)-associated leucine zipper protein (JLP) was originally identified as a binding protein for the transcription factor Max, and further biochemical study indicated that JLP functions as a scaffold protein for the JNK and p38 MAPK signalling modules (6). JLP is broadly expressed, and is structurally related to JNK/stress-activated protein kinase-associated protein 1 (JSAP1 or JNK-interacting protein 3), a scaffold protein for JNK cascades (7-9).Studies of JLP have mainly focused on identifying its interacting proteins, which include kinesin light chain 1 and G a13 , the a-subunit of the heteromeric G 13 protein (10,11). JLP has also been reported to play an important role in myogenesis by interacting with the cell-surface p...

The gep Proto-Oncogene Gα13 Mediates Lysophosphatidic Acid-Mediated Migration of Pancreatic Cancer Cells

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Regulation of Neurite Outgrowth by Interactions between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-associated Protein Superior Cervical Ganglia Clone 10

“…It appears that JLP modulates JNK-dependent phosphorylation of SCG10 and thereby NGF-induced neurite extension by sequestering JNK and/or SCG10 into a non-productive signaling complex. In fact, recent studies have shown that JLP can modulate physiological responses by sequestering active signaling components in different subcellular regions (20).…”

JNK-associated Leucine Zipper Protein Functions as a Docking Platform for Polo-like Kinase 1 and Regulation of the Associating Transcription Factor Forkhead Box Protein K1

“…One such well studied scaffolding protein family is the family of JNK-interacting proteins (JIPs). 5 The JIP family consists of four members: JIP1, JIP2, JIP3, and JLP (2). Of all the members characterized in the JIP family, JLP is characterized by the presence of two leucine zipper domains and a JNK binding domain in its N terminus and a conserved C-terminal domain, all of which functions in binding to and forming specific cell signaling complexes (3).…”

“…Of all the members characterized in the JIP family, JLP is characterized by the presence of two leucine zipper domains and a JNK binding domain in its N terminus and a conserved C-terminal domain, all of which functions in binding to and forming specific cell signaling complexes (3). Although JLP is ubiquitously expressed, previous studies have shown that the protein plays a role in regulating neurite outgrowth by interacting with SCG10 (4), activating JNK/p38 MAPK signaling (3), regulating cell migration by interacting with G␣12 and G␣13 (5,6), and functioning in vesicle transport by binding to kinesin light chain (KLC1) and dynein microtubule motor proteins (7). Recent studies have identified that JLP and JIP3 can interact with ADP-ribosylation factor 6 (ARF6), a small G protein that function in the regulating cytokinesis by transporting recycling endosomes to the cleavage furrow (8).…”