Global Phosphoproteome of HT-29 Human Colon Adenocarcinoma Cells

Abstract

Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.

Protein kinase a modulates Cdc25B activity during meiotic resumption of mouse oocytes

“…The differences in GVBD-inducing ability of the two phosphatases, Cdc25A and Cdc25B, suggest that they have different substrates and nonredundant functions (Solc et al, 2008 (Yaffe et al, 2001;Obenauer et al, 2003). This program has been quite successful and some predictions have been experimentally verified (Gratton et al, 2001;Zhou et al, 2001;Gottlieb et al, 2002;Kim et al, 2002Kim et al, , 2005She et al, 2004;Park et al, 2006;Lemeer et al, 2008). In this study, several strong PKA phosphorylation consensus sites were predicted in Cdc25B including Ser-321 which corresponds to Cdc25-S287 in Xenopus oocytes and human Cdc25B-S323.…”

Implication of Serine Residues 271, 273, and 275 in the Human Immunodeficiency Virus Type 1 Cofactor Activity of Lens Epithelium-Derived Growth Factor/p75

“…The N-terminal region of CR3, residues 267 to 275, contains six demonstrated phosphorylated Ser/Thr sites, which represent 27.2% of the identified LEDGF/ p75 Ser/Thr phosphosites (11,12,15,17,28,36,43,44). These Ser/Thr residues are predicted to be targeted by protein kinase casein kinase 2 (PKCK2) (31).…”

Interphase phosphorylation of lamin A

“…It has also been proposed that phosphorylation of Ser525 regulates either lamina assembly or its interaction with chromatin (Haas and Jost, 1993;Leukel and Jost, 1995). Tyrosine phosphorylation of lamins has also been described (Otto et al, 2001), and numerous phosphorylation sites have been identified in global, high-throughput or large-scale proteomic studies (Beausoleil et al, 2004;Kim et al, 2005;Olsen et al, 2006). However, the existence, and the functional significance, of the majority of lamin phosphorylation sites remain uncharacterized.…”