The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

The skeletal muscle specific Ca 2؉ /calmodulin-dependent protein kinase (CaMKII␤ M ) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, ␣KAP, but its function remains to be defined. Protein interactions of CaMKII␤ M indicated that it exists in complex with enzymes involved in glycolysis at the SR membrane. The kinase was found to complex with glycogen phosphorylase, glycogen debranching enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and creatine kinase in the SR membrane. CaMKII␤ M was also found to assemble with aldolase A, GAPDH, enolase, lactate dehydrogenase, creatine kinase, pyruvate kinase, and phosphorylase b kinase from the cytosolic fraction. The interacting proteins were substrates of CaMKII␤ M , and their phosphorylation was enhanced in a Ca 2؉ -and calmodulin (CaM)-dependent manner. The CaMKII␤ M could directly phosphorylate GAPDH and markedly increase (ϳ3.4-fold) its activity in a Ca 2؉ /CaMdependent manner. These data suggest that the muscle CaMKII␤ M isoform may serve to assemble the glycogenmobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKII␤ M in response to calcium signaling would serve to modulate GAPDH and thereby ATP and NADH levels at the SR membrane, which in turn will regulate calcium transport processes.Free calcium (Ca 2ϩ ) regulates diverse cellular functions by acting as an intracellular second messenger. A large part of these cellular functions are mediated by CaM, 1 which is the ubiquitous intracellular Ca 2ϩ receptor. The Ca 2ϩ ⅐CaM complex allosterically activates numerous proteins, including Ca 2ϩ /CaMdependent protein kinase II (CaMKII) (1). CaMKII is a multifunctional enzyme that is highly expressed in brain and muscle. The kinase is believed to serve important roles in synaptic transmission (2, 3), gene transcription (4, 5), cell growth (6), and control of excitation-contraction coupling (7-9).The subcellular distribution of CaMKII indicates cytosolic and membrane localizations in different tissues (10). In skeletal muscle, an isoform of CaMKII is targeted to the SR membrane by a non-kinase protein, ␣KAP (11, 12). Different studies have been conducted to determine whether membrane-bound CaM kinase could phosphorylate different substrate proteins by virtue of its proximity effects and thereby regulate SR function. Although the calcium release channel/ryanodine receptor (RyR) and the calcium pump/Ca 2ϩ -ATPase in skeletal muscle SR were shown to be substrates of CaMKII␤ (13, 14), there does not appear to be any clear effects on the regulation of functional activity of these proteins induced by such phosphorylation (7,8,(15)(16)(17). Moreover, there is clear evidence that the RyR and calcium pump are regulated by local ATP, Ca 2ϩ , and CaM through direct ligand binding (15)(16)(17). In this regard, both Ca 2ϩ and CaM are present at the SR, and the level of ATP is believed to be tightly controlled through a membrane-bound glycolytic mac...

The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with γ-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.

The sarcolemmal associated proteins (SLAPs) are encoded by multiple mRNAs that are presumably generated by alternative splicing mechanisms. The amino acid sequence of the SLAP1 isoform exhibited 76% identity with TOP AP , a topographically graded antigen of the chick visual system. The regions of coiled-coil structure including an 11-heptad acidic amphipathic ␣-helical segment was conserved with a major divergence in sequence noted in the hydrophobic C termini predicted to be transmembrane domains in the two polypeptides. The genomic organization of the 3 region of the SLAP gene indicated that SLAP1 and TOP AP are generated by alternative splicing mechanisms, which are conserved among mammalian and avian species. SLAP1/TOP AP were encoded by 11 exons distributed over a minimum of 35 kilobase pairs of continuous DNA; 9 of the exons were constitutively expressed, and 2 were alternatively spliced. The exons range in size from 60 to 321 base pairs, and the predicted functional domains within the polypeptides were encompassed by single exons. The introns vary from 0.2 to 10 kilobase pairs and conform to consensus dinucleotide splicing signals. Reverse transcriptase-polymerase chain reaction studies demonstrated that alternative exons (IV and X) of SLAP were expressed in a tissue-specific fashion and developmentally regulated. The alternatively spliced exon X, which encodes the putative transmembrane anchor in TOP AP , and a constitutively expressed exon XI, which encodes the putative transmembrane domain in SLAP, were found to target these polypeptides to membrane structures. The presence and conservation of termination codons in exons X and XI render expression of the two SLAP1/TOP AP transmembrane domains mutually exclusive. These data reveal that TOP AP and SLAP are alternatively spliced products of a single gene that encodes a unique class of tail-anchored membrane proteins.

The spatial arrangement of the cell-surface membranes (sarcolemma and transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitation-contraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique COOH-terminal membrane anchors that could target this molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac-specific expression of SLMAP isoforms that can be targeted to distinct subcellular membranes, self assemble, and interact with the myofibril suggests a potential role for this molecule in the structural arrangement of the E-C coupling apparatus.