Biomedical subjects

Len C Packman

Publications and source records attributed to Len C Packman.

5 recordsLinked to original sources

PubMed2006

The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site.

BACKGROUND: The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells. RESULTS: Overexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1. CONCLUSION: The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.

Animals↗

PubMed2004

Protein phosphatase 2A, a negative regulator of the ERK signaling pathway, is activated by tyrosine phosphorylation of putative HLA class II-associated protein I (PHAPI)/pp32 in response to the antiproliferative lectin, jacalin.

Protein phosphatase 2A (PP2A) is a family of mammalian serine/threonine phosphatases that is involved in the control of many cellular functions including those mediated by extracellular signal-regulated kinase (ERK) signaling. While investigating the reversible antiproliferative effect of the dietary lectin, jacalin, which binds the Thomsen-Friedenreich antigen (galactose beta1-3 N-acetylgalactosamine alpha-), we have found that this lectin (30 microg/ml) induces rapid, transient, tyrosine phosphorylation of putative human HLA-DR-associated protein I (PHAPI, also known as the tumor suppressor pp32) in HT29 human colon cancer cells. This is accompanied by the release of PP2A from association with PHAPI, allowing increased phosphatase activity of PP2A (by 42 +/- 10% at 10 min) and consequent complete dephosphorylation of the ERK kinase, MEK1/2, by 10 min and of ERK1/2 by 60 min. PHAPI knockdown by RNA interference abolished the effects of jacalin on PP2A activation and MEK inhibition. Thus phosphorylation of PHAPI/pp32 is a critical regulatory step in PP2A activation and ERK signaling.

HT29 Cells↗

PubMed2004

The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures.

Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual structures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccharides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual difucosylated cores. Cross-ring fragment ions and 'internal' fragment ions observed in the CID spectra, showed that these fucoses are found at the 3-position of proximal GlcNAc and at the 3-position of distal GlcNAc in the chitobiose unit. The latter substitution has only been previously observed in nematodes. In addition, these core structures can be decorated with novel fucosylated antennae composed of Fucalpha(1-3)GlcNAc. Key fragment ions revealed that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N-glycan (Man(3)GlcNAc(2)), commonly found on other insect glycoproteins, is the predominant oligosaccharide found at the remaining N-glycosylation site (Asn609).

Amino Acid Sequence↗

PubMed2003

Characterization of cyclin L2, a novel cyclin with an arginine/serine-rich domain: phosphorylation by DYRK1A and colocalization with splicing factors.

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.

Amino Acid Sequence↗

PubMed2003

ABRF-PRG03: phosphorylation site determination.

A fundamental aspect of proteomics is the analysis of post-translational modifications, of which phosphorylation is an important class. Numerous nonradioactivity-based methods have been described for high-sensitivity phosphorylation site mapping. The ABRF Proteomics Research Group has conducted a study to help determine how many laboratories are equipped to take on such projects, which methods they choose to apply, and how successful the laboratories are in implementing particular methodologies. The ABRF-PRG03 sample was distributed as a tryptic digest of a mixture of two proteins with two synthetic phosphopeptides added. Each sample contained 5 pmol of unphosphorylated protein digest, 1 pmol of each phosphopeptide from the same protein, and 200 fmol of a minor protein component. Study participants were challenged to identify the two proteins and the two phosphorylated peptides, and determine the site of phosphorylation in each peptide. Almost all respondents successfully identified the major protein component, whereas only 10% identified the minor protein component. Phosphorylation site analysis proved surprisingly difficult, with only 3 of the 54 laboratories correctly determining both sites of phosphorylation. Various strategies and instruments were applied to this task with mixed success; chromatographic separation of the peptides was clearly helpful, whereas enrichment by metal affinity chromatography met with surprisingly little success. We conclude that locating sites of phosphorylation remains a significant challenge at this level of sample abundance.

Chemistry Techniques, Analytical↗