Endocytic Trafficking and Human Diseases

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Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by mental and psychomotor retardation, diminished muscle tone or hypotonia, achlorhydria, and visual problems including corneal clouding, retinal degeneration, sensitivity to light, and strabismus. Analysis of fibroblasts from MLIV patients by electron microscopy revealed the presence of enlarged vacuolar structures that accumulate mucopolysaccharides, phospholipids, and gangliosides. These enlarged vacuoles are present not only in fibroblasts but in many different cell types, suggesting a general impairment of the lysosomal function in MLIV patients. Linkage analysis carried out in 13 Ashkenazi Jewish families mapped the MLIV locus to chromosome 19p13.3-p13.210 and allowed the identification of mucolipin-1 (MCOLN1) as the gene responsible for the disease. MCOLN1 is an ion channel with homology to the transient receptor potential (TRP) channel superfamily. The selectivity of the MCOLN1 channel remains controversial, as different studies have suggested that it is permeable to Ca2+, Ca2+, K+ and Na+, and H+. The lysosomal defects observed in MLIV patients have led to the suggestion that MCOLN1 might play a role in the biogenesis of lysosomes. Recently, several groups have suggested additional roles for MCOLN1 in different cellular processes including regulation of lysosomal acidification and lysosomal secretion. To understand better the molecular mechanisms that regulate MCOLN1 activity, we investigated whether the N- and C-terminal tails of MCOLN1 are modified by phosphorylation. Soluble proteins representing the N-terminal tail (GST-MCOLN1-NTail; residues 1-66) and the C-terminal tail (GST-MCOLN1-CTail; residues 518-580) were synthesized in bacteria as GST fusion proteins. Following purification, these fusion proteins were incubated with HeLa cell lysates and subjected to in vitro kinase assays. Our results showed that GST-MCOLN1-CTail was efficiently phosphorylated in vitro by protein kinase activities present in HeLa cells lysates. Moreover, the phosphorylation of the GST-MCOLN1-CTail chimera was almost completely abolished after treatment with staurosporine, a broad range serine/threonine kinase inhibitor, suggesting that a serine/threonine kinase present in HeLa cell lysates phosphorylates the C-terminal tail of MCOLN1. Mutagenesis analysis allowed us to identify Ser557 as the major in vitro phosphorylation site in the MCOLN1 C-tail; Ser559 is also phosphorylated but to a much lesser extent. To determine the type of serine/threonine protein kinase responsible for the phosphorylation of MCOLN1, we examined the effects of various serine/threonine protein kinase inhibitors. Using our in vitro kinase assay, we found that addition of H89 to HeLa cell lysates effectively inhibited phosphorylation. Because H89 inhibition of MCOLN1 phosphorylation is indicative of protein kinase A (PKA) activity, we sought to determine whether MCOLN1 is phosphorylated after treatment of cells with forskolin (FSK), a PKA activator. As expected, phosphorylation of MCOLN1 C-tail and MCOLN1 full-length was significantly increased after FSK treatment both in vitro and in vivo. To test the effect of MCOLN1 phosphorylation on its activity, we analyzed changes in amplitudes of MCOLN1-dependent currents associated with inhibition or activation of PKA. Interestingly, cell treatment with H89 induced an increase in amplitudes of MCOLN1-dependent currents, while treatment with FSK inhibited the currents. Based on these results we conclude that inhibition of PKA and MCOLN1 dephosphorylation increases MCOLN1 channel activity while MCOLN1 phosphorylation inhibits it. The stimulatory effect of H89 on MCOLN1 function was not observed when Ser557 and Ser559 were mutated to alanine, indicating that these two residues are essential for PKA-mediated negative regulation of MCOLN1. This study constitutes the first report of regulation of a member of the mucolipin family by phosphorylation. Sequence alignment revealed that MCOLN1 shows high homology with two other proteins named, mucolipin-2 (MCOLN2) and mucolipin-3 (MCOLN3), and the three proteins have been grouped into the mucolipin family. MCOLN3 might also play a role in different human pathologies, as mutations in this gene are responsible for the varitint-waddler mouse phenotype that is characterized by defects in pigmentation and hearing loss22. MCOLN3 is located in hair cells, and it could be implicated in hair cell maturation and melanosome trafficking. In addition we have recently described that MCOLN2 MCOLN2 traffics through the Arf6-associated pathway and regulates recycling of CD59 to the plasma membrane. Thus, our results have provided important information indicating that mucolipins are important regulators of specific intracellular trafficking events. Future experiments include the search for cellular proteins that interact with the members of the mucolipin family. We are using a combination of yeast two-hybrid screening and pull down experiments that hopefully will result in the identification of effectors that regulate MCOLNs function. In addition we have generated a battery of reagents (including rabbit polyclonal antibodies against endogenous MCOLNs, adenovirus expressing shRNA sequences that can specifically knock down each of the MCOLNs, and plasmids encoding the ORF of the three MCOLNs fused to different epitope tags, as well as chimeras carrying mutations that affect MCOLN traffic and activity) that will allow us to continue the study of the role of MCOLNs on intracellular trafficking.