👤 Jan Schepens

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5' end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3' end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts. Show less

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors. Show less