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Abstract
c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK. More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3. We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain. To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system. One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA. Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells. The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells. Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF. This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine. Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines. Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells. The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells.
Highlights
Ʈ To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Michigan Medical Center, 4570 MSRB II, Box 0650, 1150 West Medical Center Dr, Ann Arbor, MI 481090650
Identification of hJIP-1 Interacting Proteins—We first identified the phosphotyrosine binding (PTB) domain of human JNK interacting protein-1 (JIP-1) as an expressed sequence tag following a data base search for sequences similar to the PTB domain of Shc [4]
Subcellular Localization of JIP-1—In order to gain insight into the possible role that JIP-1 plays in neuronal cells, we investigated the localization of endogenous JIP-1 in N1E-115 and PC12 cells by indirect immunofluorescence
Summary
Ʈ To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Michigan Medical Center, 4570 MSRB II, Box 0650, 1150 West Medical Center Dr, Ann Arbor, MI 481090650. To further analyze the role of PTB domain proteins in cellular signaling, we have cloned and characterized the human homologue of the JNK interacting protein-1 (JIP-1). In order to further characterize the role of PTB-containing proteins in the organization of cellular signaling complexes, we sought to identify binding partners for the highly conserved PTB domain of JIP-1.
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