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Molecular and Cellular Biology, December 2007, p. 8271-8283, Vol. 27, No. 23
0270-7306/07/$08.00+0     doi:10.1128/MCB.01781-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes{triangledown}

Sanita Bharti,1 Hiroki Inoue,1 Kapil Bharti,2 Dianne S. Hirsch,3 Zhongzhen Nie,1 Hye-Young Yoon,1 Vira Artym,4,5 Kenneth M. Yamada,4 Susette C. Mueller,5 Valarie A. Barr,1 and Paul A. Randazzo1*

Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute,1 Mammalian Development Section, National Institute of Neurological Disorders and Stroke,2 Food and Drug Administration,3 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland,4 Georgetown University, Washington, DC5

Received 20 September 2006/ Returned for modification 13 November 2006/ Accepted 14 September 2007

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

* Corresponding author. Mailing address: Laboratory of Cellular and Molecular Biology, Center for Cancer Research, CCR, Building 37, Room 4118, Bethesda, MD 20892. Phone: (301) 496-3788. Fax: (301) 480-1260. E-mail: Randazzo{at}helix.nih.gov

{triangledown} Published ahead of print on 24 September 2007.

Molecular and Cellular Biology, December 2007, p. 8271-8283, Vol. 27, No. 23
0270-7306/07/$08.00+0     doi:10.1128/MCB.01781-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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