The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons

Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.     

Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells

Background

Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.

Results

We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

Conclusion

The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells.     

The association of Zygocotyle lunata and Echinostoma trivolvis with Chaetogaster limnaei, an ectosymbiont of Helisoma trivolvis

Helisoma trivolvis snails collected from a lake in Warren County, New Jersey, in June 2007 possessed the ectosymbiont Chaetogaster limnaei (Annelida). Some of these snails were also infected with larval stages of Zygocotyle lunata and Echinostoma trivolvis. Chaetogaster limnaei associated with the infected snails fed on the cercariae of both digeneans. Zygocotylae lunata cercariae were observed in the stomach of C. limnaei and whole cercariae were loosely attached to the ventral surface of the chaetogasters. Cercariae in the stomachs were digested within 48 hr and probably served as a source of nutrient for the annelids. Whole cercariae and 1 nonviable metacercaria of E. trivolvis were seen in the stomachs of the chaetogasters. The protective action of the chaetogasters on the transmission of the cercariae of E. trivolvis and Z. lunata to second intermediate hosts in the wild awaits further study.     

Comparison of theN-glycoloylneuraminic andN-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography

N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - RPM rat promegakaryoblast - MEG-01 human megakaryoblastic leukaemia cell line - PAD pulsed amperometric detection - WGA wheat germ agglutinin - FCS foetal calf serum - PPEADF phosphatidylethanolamine dipalmitoyl - LSIMS liquid secondary ion mass spectrometry - HPAEC high performance anion exchange chromatography - TBA thiobarbituric acid     

Microtubule-kinesin interface mutants reveal a site critical for communication

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.     

New mutation causing jaundice in mice

A new mutant causing jaundice in mice is reported. Allelism tests indicate that it is not allelic with known hemolytic anemia mutants in mice [hemolytic anemia (sphha), jaundiced (ja), normoblastic anemia (nb), and spherocytosis (sph and sph2Be)]. The jaundiced condition appears in young mice at about 24 hours postpartum and is due to a major increase in unconjugated bilirubin in serum compared to normal controls. Adult mutant mice are not jaundiced and bilirubin levels do not differ from normal mice. Adult male mutants have reduced testes size and no viable sperm. Female mutants are fertile but overall maternal performance is reduced as indicated by increased mortality and reduced growth rate of pups. Genetic tests indicate that a single autosomal recessive gene is responsible for the condition. We propose that the mutant be designated hyper-unconjugated bilirubinemia, with the gene symbol hub.