Karen S. Phillips

Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B)

Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.

Characterization of a rat cDNA clone encoding calcium/calmodulin-dependent protein kinase I

Two cDNA clones encoding calcium/calmodulin-dependent (CaM) protein kinase I were isolated. In contrast to the previously reported CaM kinase I cDNA, which encodes a protein with a mass of 37 kDa, the clones identified in this study encode a protein (10-1/CaM kinase I) with a predicted mass of 42 kDa; the size of 10-1/CaM kinase I was verified by hybrid-selected translation.

The testis isoform of the phosphorylase kinase catalytic subunit (PhK-T) plays a critical role in regulation of glycogen mobilization in developing lung

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-T) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-T RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-T protein expression was temporally and spatially correlated with expression of PhK-T RNA. PhK-T RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-T RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-T protein was virtually undetectable in gsd/gsd Type II cells and enzyme activity was very low. These results suggest that PhK-T plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-T RNA or unstable protein.