Analysis of the phosphoproteome of MK5-deficient tissues

Phospho-motif antibodies, which detect phosphorylated kinase-specific substrates, are a useful tool to study the mammalian phosphoproteome, allowing broad analysis and quantification of kinase-specific phosphorylation pattern. They could be helpful for identification of novel kinase substrates. So far, there is no MK substrate motif antibody available, but the putative MK phosphorylation site [FVIL]-x-R-x-x-S is similar to the basophilic motifs of the AGC kinase family members PKA, PKB, PKC, and PKD. Here, an antibody sampler kit containing antibodies raised against the PKA, PKC, CDK and ATM/ATR substrate phospho-motif (Cell Signal Technologies) was used in western blot analysis. In Tab. 9, the results of the distinct antibody staining pattern after western blotting of several tissues obtained from wild type and MK5-deficient mice are summarized. The tissues of each genotype were analyzed in triplicates, and the received staining patterns were compared.

Phosphoband [#/(kDa)] Brain Heart Kidney Liver Lung

Analysis of phospho-motif antibodies staining partially revealed differences within the same genotype. Only phospho-bands that appeared or disappeared in all three distinct lysates of the MK5-deficient tissue were taken into account (listed in the Tab.1). The resolution of the SDS PAGE was moderate, so that the number of detected phosphoproteins mostly remained below 30. Low expressed substrates or minor changes in phosphorylation could be overlooked. The strongest difference between wild type and MK5-knockout tissues has been detected in lysates of the heart using PKA/PKC phospho-motif antibodies. Additional substrates probably phosphorylated by PKA or PKC could be observed in heart tissue lacking MK5 (Fig. 23). This might indicate a negative regulation of MK5 in PKA/PKC-dependent signaling of cardiac cells. Moreover, one additional PKA phospho-substrate band has also been detected in spleen of MK5-deficient mice. The phosphoprotein migrates with a molecular mass of 110 kDa. The most convincing difference has been found in lysates of

Tab. 9. Substrate phospho-motif antibody staining pattern of PKA, PKC, CDK, and ATM/ATR kinase of MK5-deficient compared to wild type tissues showed additional (A) or missing (B) phosphoprotein-bands. Tissue lysates were generated from each three MK5-deficient and wild type mice. Additional or lost bands were only considered if all samples of specific genotype showed the same pattern.

Protein Full name cDNA

library Clone number(s) Growth Reconstitution of interaction with

baits Expressed fragment

screening /

clone# empty MK2 MK5 [aa]

1 Sept8 Septin 8 brain VI/ 4, 13, 31, 67 * fast x several ~67-484

(C-term.)

2 ATP1b1 Sodium/ potassium ATPase subunit beta 1 brain VI/1 * slow x x 204-304 (C-terminus)

3 Kal7 Rho nucleotide exchange factor kalirin 7 brain VI/ 84 slow x 588-941 (1663)

4 Mtbp Mdm2-binding protein norm.

universal IV/20 fast x 431-894 (C-term)

5 snRNP48 Small nuclear ribonucleoprotein 48 brain VI/ 25, 51, 100 slow x several ~21-271 (337)

6 Dclk1 Doublecortin-like kinase 1 brain V/53 fast x 488-756 (C-terminus)

7 Ccar Cell division cycle and apoptosis regulator

protein 1 brain VI/41 fast x x 617-839 (1146)

8 Krt222 Keratin-like protein 222 brain VI/97 slow (x) x x full-length (294)

9 CPE Carboxypeptidase E brain VI/56 slow (x) x x 176-446 (476)

10 Mtap1a Microtubule-associated protein 1a brain V/40 fast x x x 2561-2677 (C-term)

Tab. 10. Positive cDNA prey clones identified in three distinct yeast two-hybrid screens using MK5 as bait. Overall, 3.2 x 10⁶ clones of a murine brain cDNA library and 2.1 x 10⁶ clones of a mouse normalized universal cDNA library were screened and 188 clones were selected and further analyzed. Interactions with Septin8 (Sept8) and sodium pump regulatory subunit beta-1 (ATP1b1) were already known from previous screens (*), whereas interactions with the Mdm2-binding protein and the Rho nucleotide exchange factor kalirin7 (Kal7), the small nuclear ribonucleoprotein 48, the doublecortin-like kinase 1, the cell division cycle and apoptosis regulator protein 1, the keratin-like protein 222, the carboxypeptidase E, and the microtubule-associated protein 1a were unknown, so far. A gray background highlights the prey proteins, which were further analyzed in this study.

et al., 2011). Taken together, these results strongly suggest a complex role of the ERK3/MK5-signaling module in regulation of dendritic spine formation.

For direct analysis of the effect of the ERK3/MK5-signaling module on Sept7-dependent neuronal morphogenesis co-overexpression studies in mouse hippocampal neurons were performed. Neurons were isolated from embryonic day 18 BALB/c mice (E18.5) and cultivated on coated glass coverslips. After 5 days of in vitro culture (5DIV) cells were transfected with pcDNA6/BioEase-ERK3, pcDNA3.1-HA-MK5, or pRSK5-myc-Sept7 together with pEGFP to enable selection of transfected neurons. Neuronal morphology of transfected cells were analyzed three days post transfection (5+3DIV) in regard to dendrite number and branching. Neuronal cultures were fixed with 4% PFA and GFP-positive neurons were visualized using confocal microscopy. The overall complexity of statistically relevant, selected neurons transfected with pEGFP alone (CTRL) or with myc-Sept7/pEGFP without (Sept7) or together with BE-ERK3 and HA-MK5 (ERK3/MK5/Sept7) was analyzed and is shown in Fig. 43A. While Sept7 alone does not significantly increase neuronal complexity, co-expression of ERK3/MK5 leads to obviously more complex neurons possessing more dendrites with apparently more branches. The dendritic complexity was subjected to Sholl analysis. Sholl analysis counts the number of intersections made by dendrites with concentric circles of increasing distance from the cell body, reflecting both dendrite number and intersections and represents a very sensitive and objective measurement of complex branching. Performing Sholl analysis on distinct types of transfected neurons revealed that expression of Sept7, MK5, ERK3 or ERK3/MK5 together with the EGFP construct does not lead to an increase in the Sholl index (Fig. 43B). However, MK5 and ERK3 lead to a highly significant increase of counted intersections of the transfected neurons in all distances from the soma when co-expressed with Sept7.

Since Sept7 has also been described to alter dendritic spine morphology (Xie et al., 2007a), spine number in the differently transfected neurons were qualitatively analyzed. The number of spines displays a significant increase only in ERK3/MK5/Sept7 expressing dendrites (Fig. 43C). Determination of spine morphology at later stages of neuronal differentiation was also performed by transfecting primary neurons at DIV10 and inspecting morphology at DIV15 (Fig. 43D). In cells, overexpressing Sept7 alone, dendritic complexity and spine formation is rather low and did not differ significantly from control cells. Again, overexpression of Sept7 together with ERK3 and MK5 (ERK3/MK5/Sept7) leads to a dramatically increased dendritic complexity. In addition, spine formation is significantly increased in number and size in these cells (inserts in Fig. 43D).

5 Discussion

5.1 Functional aspects of ERK3/MK5-mediated signaling in MEF cells