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LAMTOR3 (MP1) and LAMTOR2 (p14) form a heterodimer as part of the larger Ragulator complex that is required for MAPK and mTOR1 signaling from late endosomes/lysosomes. Here, we show that loss of LAMTOR2 (p14) results in an unstable cytosolic monomeric pool of LAMTOR3 (MP1). Monomeric cytoplasmic LAMTOR3 is rapidly degraded in a proteasome-dependent but lysosome-independent manner. Mutational analyses indicated that the turnover of the protein is dependent on ubiquitination of several lysine residues. Similarly, other Ragulator subunits, LAMTOR1 (p18), LAMTOR4 (c7orf59), and LAMTOR5 (HBXIP), are degraded as well upon the loss of LAMTOR2. Thus the assembly of the Ragulator complex is monitored by cellular quality control systems, most likely to prevent aberrant signaling at the convergence of mTOR and MAPK caused by a defective Ragulator complex.

During normal spermatogenesis, the testis-specific linker histone H1t appears at pachytene stage becomes phosphorylated in early spermatids and disappears in late spermatids. Using reversed-phase and hydrophilic interaction liquid chromatography, H1t from rat and mouse testes was isolated, subjected to enzymatic digestion, and analyzed by mass spectrometry. We observed different phosphorylated states of H1t (mono-, di-, and triphosphorylated) as well as the unphosphorylated protein. Tandem mass spectrometry and immobilized metal ion affinity chromatography experiments with MS/MS/MS and multistage activation were utilized to identify five phosphorylation sites on H1t from rats. Phosphorylation occurs on both serine and threonine residues, whereas only two of these sites were located on peptides containing the CDK consensus motif (S/T)PXZ. Rat H1t phosphorylation starts first by phosphorylation of the nonconsensus motif SPKS in the COOH-terminal domain, namely at Ser-140 and to a smaller degree at a further nonconsensus motif at Ser-186. This is followed by phosphorylation of Ser-177 and Thr-155, both located in CDK consensus motifs. A single phosphorylation site at Ser-8 in the NH2-terminal tail was also found. Mouse H1t lacks Ser-186 and is phosphorylated at up to four sites. In contrast to somatic linker histones, no strict order of increasing phosphorylation could be detected in H1t. Thus, it appears that not the order of up-phosphorylation but the number of the phosphate groups is necessary for regulated chromatin decondensation, thus facilitating the substitution of H1t by transition proteins and protamines.

H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr137 and Thr154, the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serine- or threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Iβ. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform.

Histone H1(0) is known to consist of two subfractions named H1(0)a and H1(0)b. The present work was performed with the aim of elucidating the nature of these two subfractions. By using reversed-phase high performance liquid chromatography in combination with hydrophilic interaction liquid chromatography, we fractionated human histone H1(0) into even four subfractions. Hydrophilic interaction liquid chromatographic analysis of the peptide fragments obtained after cleavage with cyanogen bromide and digestion with chymotrypsin suggested that the four H1(0) subfractions differ only in their small N-terminal end of the H1(0) molecule (30 residues). Edman degradation of the N-terminal H1(0) peptide fragments and mass spectra analysis have indicated that human histone H1(0) consists of intact histones H1(0) (named H1(0) Asn-3) and deamidated H1(0) forms (H1(0) Asp-3) having an aspartic acid residue at position 3 instead of asparagine. Moreover, both H1(0) Asn-3 and H1(0) Asp-3 are blocked (H1(0)a Asn-3, H1(0)a Asp-3) and unblocked (H1(0)b Asn-3, H1(0)b Asp-3) on their N terminus. Acid-urea gel electrophoretic analysis has shown that the histone subfraction, in the literature originally named H1(0)a, actually consists of a mixture of H1(0)a Asn-3 and H1(0)a Asp-3, whereas H1(0)b consists of H1(0)b Asn-3 and H1(0)b Asp-3. Furthermore, we found that hydrophilic interaction liquid chromatography separates rat and mouse histone H1(0) just like human H1(0) into four subfractions. Hydrophilic interaction liquid chromatographic analysis of brain and liver histone H1(0) from rats of different ages revealed an age-dependent increase of both the N-terminally acetylated and the deamidated forms of H1(0). In addition, we found that the relative proportions of the four forms of H1(0) histones differ from tissue to tissue.