Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading.
Focal adhesion (FA) formation is induced by extracellular matrix-stimulated integrin clustering and activation of receptors for diffusible factors. Leupaxin (LPXN) is a member of the paxillin family of FA proteins expressed in many cancer cell lines. We found activation of gastrin-releasing peptide receptor (GRPr) by bombesin (BN) stimulated LPXN translocation from cytoplasm to FAs. Using mutagenesis, we identified LIM3 as the primary FA targeting domain for LPXN and showed BN-induced LPXN tyrosine phosphorylation on residues 22, 62 and 72. A LIM3 point mutant of LPXN failed to target to FAs and had no BN-stimulated tyrosine phosphorylation. Conversely, a non-phosphorylatable mutant (Y22/62/72F) translocated to FAs after BN addition. Stimulation of FA formation using vinblastine also induced LPXN translocation and tyrosine phosphorylation. Therefore, dynamic LPXN tyrosine phosphorylation requires translocation to FAs. LPXN and paxillin had opposite roles in adhesion to collagen I (CNI) in MDA-MB-231 breast cancer cells. LPXN siRNA stimulated whereas paxillin siRNA inhibited cell adhesion. Knockdown of both LPXN and paxillin behaved similarly to paxillin knockdown alone, suggesting LPXN’s function in adhesion might depend on paxillin. Additionally, LPXN regulated cell spreading on CNI but not on fibronectin whereas paxillin knockdown suppressed spreading on both substrates. These results demonstrate that although LPXN and paxillin’s FA targeting and tyrosine phosphorylation are similar, each protein has distinct functions.
Chen
Pei-Wen
PW
Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA. chenp2@mail.nih.gov
Kroog
Glenn S
GS
D000595:Amino Acid Sequence; D000818:Animals; D041702:BALB 3T3 Cells; D001839:Bombesin / Q000494:pharmacology; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D024042:Collagen Type I / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D051419:Paxillin / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014443:Tyrosine / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D024042:Collagen Type I; D016326:Extracellular Matrix Proteins; C112251:LPXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D001839:Bombesin