title: Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase.
abstract: Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors.
authors: Department of Molecular Biotechnology and Health Sciences, Università degli Studi di Torino, Torino, Italy.
authors: Daniele
authors: D
authors: Repetto
authors: Simona
authors: S
authors: Aramu
authors: Elisabetta
authors: E
authors: Boeri Erba
authors: Nanaocha
authors: N
authors: Sharma
authors: Silvia
authors: S
authors: Grasso
authors: Isabella
authors: I
authors: Russo
authors: Ole N
authors: ON
authors: Jensen
authors: Sara
authors: S
authors: Cabodi
authors: Emilia
authors: E
authors: Turco
authors: Paola
authors: P
authors: Di Stefano
authors: Paola
authors: P
authors: Defilippi
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C532615:SRCIN1 protein, human; D014443:Tyrosine; D000081247:CSK Tyrosine-Protein Kinase; D016315:Proto-Oncogene Proteins c-abl; D019061:src-Family Kinases; C000631346:CSK protein, human
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