Src-class kinases act within the agrin/MuSK pathway to regulate acetylcholine receptor phosphorylation, cytoskeletal anchoring, and clustering.

Synaptogenesis at the neuromuscular junction requires agrin-induced stable localization of acetylcholine receptors (AChRs) at the endplate. The effects of agrin are transduced by the muscle-specific receptor tyrosine kinase (MuSK). This study provides evidence that Src-class protein tyrosine kinases mediate the effects of agrin-activated MuSK to regulate clustering and anchoring of AChRs in skeletal muscle. MuSK was complexed with both Src and Fyn in the C2 mouse muscle cell line. These associations were enhanced by agrin and by increasing protein tyrosine phosphorylation with pervanadate. Coupling between MuSK and the Src-class kinases in vivo appeared to be caused by a phosphotyrosine-SH2 domain interaction because binding of MuSK to the SH2 domains of Fyn and Src in vitro was specific, enhanced by phosphorylation, and dependent on MuSK autophosphorylation. In addition, Src and Fyn phosphorylated MuSK. AChR phosphorylation, stimulated by agrin or pervanadate, was inhibited by blocking Src-class kinases with PP1. Furthermore, agrin-induced clustering and cytoskeletal anchoring of AChRs was dependent on Src-family kinases. These data support the conclusion that Fyn and Src act downstream of MuSK to regulate the stable localization of AChRs at the neuromuscular endplate during agrin-induced synaptogenesis.

Quantitative analysis from CT is prognostic for local control of supraglottic carcinoma.

BACKGROUND: The purpose of this study was to determine whether pretreatment imaging with CT was prognostic for control of the primary site in patients with squamous cell carcinoma of the supraglottic larynx. METHODS: Pretreatment CT studies were obtained on 28 patients treated definitively with radiation therapy for supraglottic larynx cancer between 1991 and 1997. Follow-up ranged from 20-58 months. RESULTS: Local control was achieved in 61% of patients. Tumor volumes ranged from 0-68.6 cm(3), with a median of 3.1 cm(3). Local control rates for tumors with volumes greater than or less than 8 cm(3) were 20% and 70%, respectively (p =.0077). Mean tumor volumes for patients with and without recurrences were 10 cm(3) and 3.4 cm(3), respectively. CONCLUSIONS: This study demonstrates that quantitative analysis from CT imaging is prognostic for control of the primary site when radiation therapy is given for treatment of supraglottic cancer.

Guanine nucleotide transport by atractyloside-sensitive and -insensitive carriers in isolated heart mitochondria.

In previous work (McKee EE, Bentley AT, Smith RM Jr, and Ciaccio CE, Biochem Biophys Res Commun 257: 466-472, 1999), the transport of guanine nucleotides into the matrix of intact isolated heart mitochondria was demonstrated. In this study, the time course and mechanisms of guanine nucleotide transport are characterized. Two distinct mechanisms of transport were found to be capable of moving guanine nucleotides across the inner membrane. The first carrier was saturable, displayed temperature dependence, preferred GDP to GTP, and did not transport GMP or IMP. When incubated in the absence of exogenous ATP, this carrier had a V(max) of 946 +/- 53 pmol. mg(-1). min(-1) with a K(m) of 2.9 +/- 0.3 mM for GDP. However, transport of GTP and GDP on this carrier was completely inhibited by physiological concentrations of ATP, suggesting that this carrier was not involved with guanine nucleotide transport in vivo. Because transport on this carrier was also inhibited by atractyloside, this carrier was consistent with the well-characterized ATP/ADP translocase. The second mechanism of guanine nucleotide uptake was insensitive to atractyloside, displayed temperature dependence, and was capable of transporting GMP, GDP, and GTP at approximately equal rates but did not transport IMP, guanine, or guanosine. GTP transport via this mechanism was slow, with a V(max) of 48.7 +/- 1.4 pmol. mg(-1). min(-1) and a K(m) = 4.4 +/- 0.4 mM. However, because the requirement for guanine nucleotide transport is low in nondividing tissues such as the heart, this transport process is nevertheless sufficient to account for the matrix uptake of guanine nucleotides and may represent the physiological mechanism of transport.

Identification of a dynein molecular motor component in Torpedo electroplax; binding and phosphorylation of Tctex-1 by Fyn.

The microtubule protein Tctex-1 was cloned from Torpedo electroplax, a biochemical model of the neuromuscular junction, using the unique domain of Fyn in the yeast two hybrid system. Binding of Tctex-1 and Fyn also occurred in vitro. Torpedo Tctex-1 was contained within the molecular motor protein dynein. A Src class kinase was also complexed with dynein. Tctex-1 was enriched in electric organ vs. skeletal muscle, was present in the postsynaptic membrane, and coprecipitated with the acetylcholine receptor. The sequence of Tctex-1 contained a tyrosine phosphorylation motif and Tctex-1 could be phosphorylated by Fyn in vitro and in vivo. These data demonstrated that Tctex-1-containing dynein is a cytoskeletal element at the acetylcholine receptor-enriched postsynaptic membrane and suggested that Tctex-1 may be a substrate for Fyn.