Tyrosine and serine phosphorylation regulate the conformation and subsequent threonine phosphorylation of the L1 cytoplasmic domain

Abstract

Previously we identified threonine-1172 (T1172) in the cytoplasmic domain of the cell adhesion molecule L1 as phosphorylated in pancreatic cancer cells. Although both CKII-and PKC-blockade suppressed this modification, only CKII was capable of phosphorylating T1172 of a recombinant L1 cytoplamic domain, suggesting the requirement for additional events to facilitate availability of T1172 to PKC. In this study, we demonstrate that the region around T1172 exists in distinct conformations based on both T1172 phosphorylation and the integrity of surrounding residues. We further demonstrate the role of membrane-proximal and membrane-distal residues in regulating cytoplasmic domain conformation, and that modification of 3 of the 4 tyrosines in the L1 cytoplasmic domain promote conformational changes that facilitate other events. In particular, phenylalaninesubstitution of tyrosine-1151 or tyrosine-1229 promote opening up of the cytoplasmic domain in a manner that facilitates phosphorylation of the other 3 tyrosines, as well as phosphorylation of T1172 by PKCα. Importantly, we show that phosphorylation of serine-1181 is required for T1172 phosphorylation by CKII. These data define a specific role for secondary structure in regulating the availability of T1172 that facilitates phosphorylation by PKC.

Modification of the L1-CAM carboxy-terminus in pancreatic adenocarcinoma cells

“…Indeed, in this report, we show that C20 reactivity reflects the presence of a distinct conformation of the L1 ectodomain, consistent with prior reports of L1 ectodomain conformation regulation by cytoplasmic domain modification [19]. While we recognize the limitations of our studies utilizing recombinant proteins, these data are consistent with our previous observations on the flexibility of the L1 ectodomain and cytoplasmic domain, and the inter-regulated nature of each [19, 29]. We further relate our recombinant in vitro data to studies of endogenous L1 and L1 chimeras expressed in PDAC cells.…”

Inside-Out Regulation of L1 Conformation, Integrin Binding, Proteolysis, and Concomitant Cell Migration

“…Although cellular phosphorylation of Y1176 and S1181 has been demonstrated previously ( Wong et al , 1996 ; Schaefer et al , 2002 ), T1172 phosphorylation has not. Previously, we demonstrated that mutation of Y1176 and S1181 does not affect 2C2 binding ( Chen et al , 2009 ); however, a size-enhancing (T1172F) or negative charge-imparting (T1172E) mutation at 1172 abrogated 2C2 binding, whereas alanine substitution had only partial effect ( Chen et al , 2009 ). Thus, although T1172 may not be an integral component of the 2C2 epitope, our data indicate that the addition of a large, charged phosphate to T1172 would prevent 2C2 binding to L1.…”

“…Rabbit serum was affinity depleted of nonphospho-T1172-dependent species on a nonphospho-KDETFGE column, and nonbound antibody was further purified on a phospho-KDETFGE column. Antibody eluted from the nonphospho-T1172 column (αT1172-IND) recognizes the KDETFGE sequence in a manner independently of T1172 phosphorylation (i.e., it demonstrates nearly equal signal on phospho- and nonphospho-peptides); the αP-T1172 species purified from the KDEpTFGE column demonstrates specific recognition of the phosphorylated peptide ( Chen et al , 2009 ). αErk2 pAb (C14), αGST pAb (110-218), and a 6xHis tag-specific pAb (H3) were from Santa Cruz Biotechnology.…”

“…A separate mass spectroscopy analysis does suggest potential phosphorylation of T1247 in L1 signal obtained from the cytoplasm and nucleus of HeLa cells ( Olsen et al , 2006 ); however, this has yet to be corroborated by other means or investigated in other cells. Previously, we demonstrated that a novel threonine in the L1 CD, immediately upstream of the alternatively spliced neuronal exon27, can be phosphorylated in vitro ( Chen et al , 2009 ). Here, we report that this residue exhibits steady-state saturated phosphorylation in PDAC cells, an event regulated by casein kinase II (CKII), protein kinase C (PKC) and protein phosphatase (PP)1, and that is associated with the regulation of L1 ectodomain (ECD) conformation, integrin-binding and a disintegrin and metalloproteinase (ADAM)-mediated proteolysis, and the concomitant control of cell migration in a matrix-specific manner.…”

An ankyrin-binding motif regulates nuclear levels of L1-type neuroglian and expression of the oncogene Myc in Drosophila neurons

“…Phosphorylation has been well-documented to regulate nuclear translocation and transcription activation (34). A prior study suggests that the ICD of L1CAM exists in an open and folded conformation and that phosphorylation of Tyr-1151 or Tyr-1229 within the FERM domain and FIGQY motif promotes opening of the cytoplasmic domain to mediate inside-out signaling of L1CAM (36). Therefore, phosphorylation of the FIGQY motif may have an indirect role in regulating nuclear levels of Nrg180 by inducing a conformation change of the intracellular domain as a mechanism to unmask motifs that regulate nuclear import or export.…”