Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility.

Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that alpha4 and alpha5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either alpha4beta1- or alpha5beta1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that alpha5beta1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for alpha5beta1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, alpha4beta1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatase-alpha overexpression inhibited alpha4beta1-stimulated NB motility and Src activation consistent with alpha4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In alpha4 shRNA-expressing NB cells, alpha4beta1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated alpha4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that alpha4beta1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during alpha5beta1-mediated NB migration and support the evaluation of inhibitors to alpha4, Src and FAK in the control of NB tumor progression.

Highly divergent actin expressed in a Chlamydomonas mutant lacking the conventional actin gene.

The Chlamydomonas mutant ida5 is deficient in the conventional actin gene and its axoneme lacks a subset of inner dynein arms that contain actin as a subunit. However, this mutant retains some other inner dynein arms because a novel protein (NAP) is expressed as a substitute for actin. In this study, we show by sequence analysis that NAP is identical to a putative actin-related protein, the cDNA sequence of which has recently been reported and shown to have 64% amino acid identity with conventional actin. A polyclonal antibody raised against a synthetic polypeptide corresponding to the NH2-terminal sequence of this protein specifically reacted with the spot corresponding to NAP in two-dimensional electrophoresis patterns. NAP apparently can substitute for conventional actin in some, but not all, cellular functions, and therefore can be regarded as a highly divergent actin. This unconventional actin appears to be expressed only when conventional actin is absent.