The instructive precedent for galectin phosphorylation is hu
The instructive precedent for galectin phosphorylation is human galectin-3 (hGal-3). Its serine modification by protein kinase CK1 in the N-terminal tail is relevant for nuclear export during the course of apoptosis regulation and contact formation with the neural cell adhesion molecule L1 , , , . The N-terminal peptide region of this chimera-type (tridomain) galectin is constituted by the tail part and collagen-like repeats besides the canonical carbohydrate recognition domain (CRD). Proving conservation of substrate properties in phylogenesis, the same sequence motif is phosphorylated in chicken Gal-3 (CG-3) . Despite the presence of two consensus sites for CK1 in the CRD, no further phosphorylation occurred in Gal-3 , . Except for Gal-3, the aspect of phosphorylation has not been studied for other members of the galectin family, as was ascertained from cross-checking of large-scale phosphoproteomics studies (). Therefore, it is an open question whether the CRDs of other galectins are substrates for Ser/Thr (S/T)-dependent kinases. Moreover, the given examples for multifunctionality of galectins give reason to study galectin phosphorylation.
To address this question, we first searched for common phosphorylation consensus motifs. We examined galectins from all three groups of topological CRD presentation: homodimeric (proto-type) Gal-1, -2, and -7/monomeric Gal-5; chimera-type Gal-3; tandem repeat-type Gal-4, -8, and -9. Encouraged by numerous hits, the galectins were tested by assays using the kinases CK1, CK2, and PKA, respectively. Phosphorylation was traced by autoradiography (ARG)/phosphoprotein staining. The combined use of in-gel digestion, phosphopeptide enrichment, and tandem mass spectrometry (MS/MS) was employed for phosphosite identification. Corresponding peptide pairs from unmodified and phosphorylated hydrazide were used to estimate the site-specific degree of phosphorylation by the ratios of the signal intensities. Finally, the position of the detected sites was visualized within the galectins. Our results reveal that the CRD of several galectins can be phosphorylated, with marked differences even between closely related members of the galectin family.
Materials and methods
Results and discussion
Conclusions This article positively answers the question of whether galectins have substrate properties for S/T protein kinases in their CRDs. A marked selectivity of phosphorylation between members of a given subgroup and species was observed. The differences within the measured profiles argue for functional relevance, as noted for serine phosphorylation in the cytosolic segments of the hepatic asialoglycoprotein receptor, the molecular chaperone calnexin, and l-selectin, all integral membrane proteins , , , , . Remarkable dynamics of phosphate removal from lectins (e.g., by protein phosphatase 2A in l-selectin)  and our current results, thus, prompt looking for the presence of phosphorylated galectins in vitro and in vivo. Galectins share the ability for multitasking with a series of other proteins , , ; therefore, glycan binding is not the sole function of galectins. With regard to possible interaction sites with other types of ligands, such as oncogenic H-Ras and the pre-B cell receptor for Gal-1 , , the profiles of phosphorylation sites will help to explain their functional impact of phosphorylation. Such a constellation would argue against a nonfunctional or accidental modification . Galectin phosphorylation may have a distinct function in fine-tuning, affecting certain tasks of the respective galectins. Equally important, the established analytical strategy can be applied to investigate phosphorylation by other protein kinases (e.g., specific for tyrosine residues).
Introduction One of the promising applications of stem cell research is the generation of cardiomyocytes for tissue replacement therapy in patients with ischemic heart disease. Since a large number of cardiomyocytes are lost during myocardial infarction, highly efficient differentiation approaches to derive cardiomyocytes from human embryonic stem cells (hESC) are required for therapeutic intervention . Although hESCs have the ability to spontaneously differentiate into cardiomyocytes in suspension culture, this process is inefficient and generates numerous cell types of other lineages. Consequently researchers have investigated the use of novel directed differentiation protocols involving the timed application of biomolecules and small molecule inhibitors as a strategy to increase the yield and purity of cardiomyocytes from hESCs .