LIS1 IS A MICROTUBULE-ASSOCIATED PHOSPHOPROTEIN

Lissencephaly, a severe brain malformation, may be caused by mutations in the LIS1 gene. LIS1 encodes amicrotubule-associated protein (MAP) that is also part of the enzyme complex, platelet-activating factoracetylhydrolase. LIS1 is also found in a complex with two protein kinases; a T-cell Tat-associated kinase, whichcontains casein-dependant kinase (CDK) activating kinase (CAK), as well as CAK-inducing activity, and with aspleen protein-tyrosine kinase similar to the catalytic domain of p72syk. As phosphorylation is one of the ways tocontrol cellular localization and protein–protein interactions, we investigated whether LIS1 undergoes this post-translational modification. Our results demonstrate that LIS1 is a developmentally regulated phosphoprotein.Phosphorylated LIS1 is mainly found in the MAP fraction. Phosphoamino acid analysis revealed that LIS1 isphosphorylated on serine residues. Alkaline phosphatase treatment reduced the number of visible LIS1 isoforms.In-gel assays demonstrate a 50-kDa LIS1 kinase that is enriched in microtubule-associated fractions. In vitro,LIS1 was phosphorylated by protein kinase CKII (casein kinase II), but not many other kinases that were tested.We suggest that LIS1 activity may be regulated by phosphorylation.Keywords: lissencephaly; LIS1; microtubules; phosphorylation.The phosphorylation state of many proteins is modulated duringdevelopment of the nervous system. Cytoskeletal rearrange-ment is one of the important processes involved in theformation of brain structure. Microtubules and microtubule-associated proteins (MAPs) are major components of the neuralcytoskeleton. The phosphorylation state of MAPs plays apredominant role in regulating their ability to bind to andstabilize microtubules in vitro and in vivo. In general, it hasbeen shown that there is an inverse correlation between phos-phorylation and microtubule binding; the more phosphorylatedthe protein, the less it binds to microtubules and stabilizes them[1–3]. The phosphorylation state of MAPs changes duringdevelopment (reviewed in [4,5]). This may be due to combinedactivities of protein kinases or protein phosphatases [6]. Thispost-translational modification is believed to be the majormechanism by which MAPs function is regulated.Lissencephaly is a severe brain malformation occurring inhumans, where the cortex is devoid of most gyri (agyria)with shallow sulci (pachygyria) [7,8]. Cerebral cortexstructure differs in patients with lissencephaly, exhibiting afour layer structure (in contrast to the normal six layerstructure) composed of neurons that are abnormally positioned.Lissencephaly is therefore categorized as an abnormal neuronalmigration defect [9–11]. We have cloned LIS1 as the genedefective in lissencephaly type I patients [12]. Human LIS1 andmurine Lis1 are expressed in the developing brain in regionsundergoing migration, both in the cortex and the cerebellum[13]. Our previous work has demonstrated that LIS1 binds tomicrotubules and affects microtubule dynamics by suppressingcatastrophe events [14]. MAPs can be classified into twocategories: structural MAPs and motility related MAPs (motorproteins). The microtubule binding motifs of structural MAPshave been characterized in detail. The microtubule bindingdomain of type 1 MAPs (MAP1A and MAP1B, as prototypes)is composed of several irregularly spaced imperfect amino acidrepeats (KKEX) in addition to light chain binding sites in theN-terminal region of the molecules. Type 2 MAPs (MAP2a/b,MAP4, Tau, PNS-Tau, and MAP2c) bind microtubules via threeor four imperfect 18 amino acid repeats at the C-terminalportion of the polypeptide chain. MAP binding to microtubulesrequires ATP hydrolysis and is best characterized for thekinesin family and cytoplasmic dynein [15]. During the recentyears several unconventional MAPs have been identifiedserving different cellular functions in addition to or incombination with their tubulin binding properties. LIS1 is anunconventional MAP, as it lacks the conventional binding sitestypical of classical MAPs and is also part of an enzymaticcomplex of platelet-activating factor acetylhydrolase [16].Evolutionary conservation of the LIS1 protein suggestsfunctional conservation [17]. Indeed a LIS1 homolog wasisolated from Aspergillus nidulans, where it is required formicrotubule-dependent nuclear migration [18].Interest in LIS1 phosphorylation resulted from its possibleinteractions with protein kinases. We have previously charac-terized a T-cell Tat-associated kinase (TTK), which containedCDK activating kinase (CAK), as well as CAK-inducing