Phosphorylation and Dephosphorylation in the Proline-Rich C-Terminal Domain of Microtubule-Associated Protein 2 (original) (raw)

1996, European Journal of Biochemistry

The C-terminal domain of microtubule-associated protein 2 (MAP2) contains a proline-rich region and the tubulin-binding domain. We have generated antibodies to follow the phosphorylation state of the proline-rich domain. One of these antibodies (no. 305) has been raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the threonine residues. This sequence is present in the proline-rich region of MAP2 and is phosphorylated in vitro by at least three different proline-directed protein kinases : p3PdC2, and GSK3 (glycogen-synthase kinase 3) alp. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as determined by Staphylococcus aureus V8 protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all three kinases studied with similar efficiency. In high-molecular-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PPl) and 2A (PP2A) catalytic subunits but not by PP2B. Keywords : microtubule ; microtubule-associated protein 2 ; proline-directed protein kinase ; serinelthreonine protein phosphatase. Neuronal microtubules are composed of the core protein tubulin and several microtubule-associated proteins. One of the most abundant microtubule-associated proteins within brain is microtubule-associated protein 2 (MAP2). This molecule has multiple isoforms generated from a single gene through alternative RNA splicing. MAP2 isoforms are divided into high-molecular-mass MAP2 proteins, which include MAP2A (consisting of 1912 amino acids in rat, with an apparent molecular mass of 280 kDa) [I] and MAP2B (1830 amino acids and an apparent molecular mass of 270kDa) [2], and low-molecular-mass MAP2, MAP2C, and MAP2D (consisting of 467 and 498 amino acids, respectively, with apparent molecular masses in the range 70-75 kDa) [3,4]. Low-molecular-mass MAP2 contains the Nand C-terminal domains of high-molecular-mass MAP2 linked together, and lacks the middle intervening sequence. Recently, additional isoforms have been found [5, 61. The location and developmental expression of these isoforms are different. MAP2B and MAP2C are expressed during early fetal development. MAP2C is down-regulated postnatally with a concomitant increase in the expression of MAP2A [7, 81. MAP2D expression increases at a later stage of development [3]. Whereas MAP2C is present in neuronal cell bodies, dendrites and axons, as well as in glial cells [9, 101, high-molecular