PhosphoregDB: the tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

Abstract

Background: Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse http://phosphoreg.imb.uq.edu.au that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse.

Results: The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases.

Conclusion: Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered.

Figure 1

Experimental localization. i) Rapid PCR generation of tagged expression constructs. A linear expression construct is produced by fusing three fragments: a CMV promoter, a dual SV40 terminator and the myc tagged open reading frame to be tested. The epitope tag is added by use of a gene specific primer that fuses the epitope in-frame with the open reading frame. Overlap sequences corresponding to AL41/GSN1 and P15/AL25 are used to prime off each other to generate a full length construct consisting of all three products. Nested primers AL35 and AL38 are used to further amplify the construct. Primer sequences and C-terminal schematics available as Supplemental data. ii) Representative observed localizations. A) Cytoplasmic: (left to right) Rps6kb1, 4932415A06Rik, Camk2d, Lats2 B) Ubiquitous, nuclear and cytoplasmic: Csnk1a1, Camkk2, Stk35, Mapkapk3 C) Nuclear: Dusp4, Mastl, Smok1, Trib1 D) Membrane associated: Ptpn5, Txk, Ptp4a3, Vrk2. Bar 10 μm.

Figure 2

Sub-cellular distributions of mouse protein kinases and phosphatases.

Figure 3

Protein kinases and phosphatases display tissue specificity. Hierarchical clustering of the expression patterns of 571 kinases and phosphatases across the 61 tissues of the GNF gene atlas. Genes and tissues were clustered in Genespring 6.2 using Pearson correlation.

Figure 4

Tissue restriction of protein kinases and phosphatases. Counts were made of the number of tissues a given gene was detected in (above 200). Then genes were divided into bins of size 10, where bin 1–10 corresponded to genes detected in 10 or fewer tissues. Bin 61, corresponds to the fraction detected in all 61 tissues.

Figure 5

Correlation of class and localization with tissue specificity. Using the same bins as described in figure 4 we demonstrate the relationship between classification and sub-cellular localization with tissue specificity. A) Serine/threonine kinases (STK n = 278), Tyrosine kinases (YK n = 79), serine/threonine phosphatases (PPM/PPP n = 25) and dual specificity/tyrosine phosphatases (DUSP/YP n = 67) B) Nuclear (n = 80), Cytoplasmic (n = 153), Nucleo-cytoplasmic (n = 106), Plasma membrane (n = 74).

Figure 6

PhosphoregDB interface. A) Example entry for Ppp1cc B) Advanced Query interface demonstrating a search for Tyrosine kinases, localizing to the nucleus and expressed in testis C) Results of a batch search for "membrane associated" phosphoregulators with experimental support.