PhosphoSitePlus, 2014: mutations, PTMs and recalibrations

Abstract

PhosphoSitePlus(®) (PSP, http://www.phosphosite.org/), a knowledgebase dedicated to mammalian post-translational modifications (PTMs), contains over 330,000 non-redundant PTMs, including phospho, acetyl, ubiquityl and methyl groups. Over 95% of the sites are from mass spectrometry (MS) experiments. In order to improve data reliability, early MS data have been reanalyzed, applying a common standard of analysis across over 1,000,000 spectra. Site assignments with P > 0.05 were filtered out. Two new downloads are available from PSP. The 'Regulatory sites' dataset includes curated information about modification sites that regulate downstream cellular processes, molecular functions and protein-protein interactions. The 'PTMVar' dataset, an intersect of missense mutations and PTMs from PSP, identifies over 25,000 PTMVars (PTMs Impacted by Variants) that can rewire signaling pathways. The PTMVar data include missense mutations from UniPROTKB, TCGA and other sources that cause over 2000 diseases or syndromes (MIM) and polymorphisms, or are associated with hundreds of cancers. PTMVars include 18 548 phosphorlyation sites, 3412 ubiquitylation sites, 2316 acetylation sites, 685 methylation sites and 245 succinylation sites.

Figure 1.

Downloads available from PSP. (1) The results pages of user-initiated searches for proteins and sites can be imported with the download button at the top of the page. (2) Static Downloads (www.phosphosite.org/staticDownloads.do) are updated monthly. The ‘Phosphosite_seq’ download contains all sequences of proteins currently in PSP (FASTA); All site datasets provide information including names and UIDs, modsite sequence, chromosomal location, Site Group ID, the number of HTP and LTP records, etc.; Disease-associated sites all have curated evidence linking them directly to a disease; BioPAX and Plugin provide pathway informatics extracted from PSP; ‘Kinase Substrate’, ‘PTM-VAR’ and ‘Regulatory sites’ all described within the text.

Figure 2.

The interactive popup page of the Cytoscape Plugin for importing data from PSP into Cytoscape.

Figure 3.

Comparison of the % of amino acid divergence (Y axis) of phosphorylation sites (pSer/pThr/pTyr) with varying numbers of associated hits (X axis). Ser, Thr or Tyr variants were excluded from counting. A total of 203,161 human/mouse and 148,441 mouse/rat phosphorylation sites were compared. The inset indicates the number of phosphosites included in each comparison. Blue, fraction of amino acid substitutions between human phosphosites and orthologous mouse residues; red, fraction of amino acid substitutions between mouse phosphosites and orthologous rat residues.

Figure 4.

Classification of modification sites in the PTMVar dataset. Class I PTMVars are those in which a site is lost by an amino acid substitution of the modified residue. The variant in this case, PAH S16P (VAR_000869), causes phenylketonuria (2). Class Ia PTMVars are those in which the variant AA can still be enzymatically modified with the same side group as the wt substrate. The variant in this case, TIE2 Y897S (VAR_008716), is associated with venous malformations (VMCM; OMIM 600195) (29). Class II PTMVars are those in which the mutation occurs on a flanking residues ±5 amino acids from the modification site. The variant in this case, PKAR1α R74C (VAR_046895), is associated with Carney complex (CNC), a familial multiple neoplasia syndrome (30).

Figure 5.

Sequence logos of 10 different kinases from various kinase groups. The number of substrate sequences used to generate each logo: SRC, 505; Akt, 200; GRK2, 58; ATM, 177; PKCα, 472; MAPKAPK2, 53; PIM1, 26; ERK2, 338; CK2-α1, 506 and PAK1, 63. Logos generated at www.phosphosite.org/siteSearchSelectAction.do using the Frequency Change logo graph method (31).

Figure 6.

Sequence logos generated using increasing numbers of input sequences. At 5 input Akt sequences, the dominance of R at −3 and −5 was already evident. The preference of PKCα for R or K at +2 was evident using 10 input sequences, but the preference for R at −2 and −3 required between 10 and 50 input sequences. Logos were generated using the Frequency Change algorithm at http://www.phosphosite.org/sequenceLogoAction.do.

Figure 7.

The locations of six types of PTM relative to Pfam-A domains. The modification types and number of modsites included in each group are: Me: 1- and 2-methyl-Arg, 3189;pS: phospho-Ser, 96570;pT: phospho-Thr, 40358;pY: phospho-Tyr, 31948;Ac: acetyl-Lys, 7403;Ubi: ubiquityl-Lys,17397.